Pyrrolobenzodiazepines

ABSTRACT

Pyrrolobenzodiazepine (PBDs) having a (1-methyl-1H-pyrrol-3-yl)phenyl based amino residue were found to be highly effective compounds having improved cytotoxicity ad DNA binding properties.

The present invention relates to pyrrolobenzodiazepines (PBDs) and inparticular to PBD monomers with a 4-(1-methyl-1H-pyrrol-3-yl)benzylbased amino acid residue containing substituent and methods ofsynthesising PBD monomers.

BACKGROUND TO THE INVENTION

Some pyrrolobenzodiazepines (PBDs) have the ability to recognise andbond to specific sequences of DNA; the preferred sequence is PuGPu. Thefirst PBD antitumour antibiotic, anthramycin, was discovered in 1965(Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965);Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Sincethen, a number of naturally occurring PBDs have been reported, and over10 synthetic routes have been developed to a variety of analogues(Thurston, et al., Chem. Rev. 1994, 433-465 (1994)). Family membersinclude abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148(1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206(1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem.Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758(1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667(1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29,93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41,1366-1373 (1988)), prothracarcin (Shimizu, et al, J. Antibiotics, 35,972-978 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97 (1987)),sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704 (1988);Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin (Leber,et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin (Arima,et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of the generalstructure:

They differ in the number, type and position of substituents, in boththeir aromatic A rings and pyrrolo C rings, and in the degree ofsaturation of the C ring. In the B-ring there is either an imine (N═C),a carbinolamine (NH—CH(OH)), or a carbinolamine methyl ether(NH—CH(OMe)) at the N10-C11 position which is the electrophilic centreresponsible for alkylating DNA. All of the known natural products havean (S)-configuration at the chiral C11a position which provides themwith a right-handed twist when viewed from the C ring towards the Aring. This gives them the appropriate three-dimensional shape forisohelicity with the minor groove of B-form DNA, leading to a snug fitat the binding site (Kohn, In Antibiotics III. Springer-Verlag, NewYork, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc. Chem. Res.,19, 230-237 (1986)). Their ability to form an adduct in the minorgroove, enables them to interfere with DNA processing, hence their useas antitumour agents. The synthesis of the compounds has been reviewedin Thurston, D. E., et al., Chem. Rev., 1994, 94, 433-465 and Thurston,D. E., et al., Chem. Rev., 2011, 111, 2815-2864.

A number of conjugates of PBD with pyrroles and imidazoles have beenreported, such as:

where n=1-3 (Damayanthi, Y., et al., Journal of Organic Chemistry,64(1), 290-292 (1999));

where n=1-3 and

where n=1-2 (Kumar, R. and Lown, J. W. Oncology Research, 13(4), 221-233(2003)); Kumar, R., et al., Heterocyclic Communications, 8(1), 19-26(2002));

where n=1-4, (Baraldi, P. G., et al., Journal of Medicinal Chemistry,42(25), 5131-5141 (1999));

where n=3, (Wells, G., et al., Proc. Am. Assoc. Canc. Res., 2003, 44,452).

In WO 2007/039752 and Wells, G, et al., Journal of Medicinal Chemistry2006, 49, 5442-5461, the following compound (GWL-78)

and related structures were disclosed in work by some of the presentinventors. This compound showed an up to 50-fold increase in DNA bindingaffinity compared to its constituent PBD and dipyrrole components.

In WO 2005/085177, some of the present inventors disclosed amino acidscomprising a biaryl core that could have useful properties in DNAbinding.

The inventors have now discovered that the properties, particularlycytoxicity and DNA binding, of the prior art PBD conjugates can beimproved. In particular, the present invention relates to theincorporation of a single 4-(1-methyl-1H-pyrrol-3-yl)benzyl based aminoacid residue in combination with a single heteroaryl based amino acidresidue in a PBD conjugate results in highly effective compounds.

A first aspect of the present invention provides a compound of formulaI:

or a salt or solvate thereof, wherein:the dotted double bond indicates the presence of a single or double bondbetween C2 and C3;R² is selected from —H, —OH, ═O, ═CH₂, —CN, —R, OR, halo, dihalo, ═CHR,═CHRR′, —O—SO₂—R, CO₂R and COR;R⁷ is selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′, nitro, Me₃Snand halo; where R and R′ are independently selected from optionallysubstituted C₁₋₇ alkyl, C₃₋₂₀ heterocycyl and C₅₋₂₀ aryl groups;R¹⁰ and R¹¹ either together form a double bond, or are selected from Hand QR^(Q) respectively, where Q is selected from O, S and NH and R^(Q)is H or C₁₋₇ alkyl or H and SO_(x)M, where x is 2 or 3, and M is amonovalent pharmaceutically acceptable cation;A is either:

where X and Y are selected from: CH and NMe; COH and NMe; CH and S; Nand NMe;

N and S;

B is either a single bond or

where X and Y are as defined above; andR¹ is C₁₋₄ alkyl.

Thus, B1 can have the following structures:

X Y B1 CH NMe

COH NMe

CH S

N NMe

N S

A second aspect of the present invention provides a method of synthesisof a compound of formula I.

A third aspect of the present invention provides a pharmaceuticalcomposition comprising a compound of the first aspect of the inventionand a pharmaceutically acceptable carrier or diluent.

A fourth aspect of the present invention provides a compound of thefirst aspect of the invention for use in a method of therapy.

A fifth aspect of the present invention provides the use of a compoundof the first aspect of the invention in the manufacture of a medicamentfor the treatment of a proliferative disease. This aspect also providesa compound of the first aspect for use in a method of treatment of aproliferative disease.

A sixth aspect of the present invention provides a method of treatmentof a patient suffering from a proliferative disease, comprisingadministering to said patient a therapeutically acceptable amount of acompound of the first aspect or a composition of the third aspect.

In the fourth to sixth aspects of the invention, the compound of theinvention may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated. In the third aspect of the invention, thepharmaceutical composition may comprise one or more (e.g. two, three orfour) further active agents.

DEFINITIONS Substituents

The phrase “optionally substituted” as used herein, pertains to a parentgroup which may be unsubstituted or which may be substituted.

Unless otherwise specified, the term “substituted” as used herein,pertains to a parent group which bears one or more substitutents. Theterm “substituent” is used herein in the conventional sense and refersto a chemical moiety which is covalently attached to, or if appropriate,fused to, a parent group. A wide variety of substituents are well known,and methods for their formation and introduction into a variety ofparent groups are also well known.

Examples of substituents are described in more detail below.

C₁₋₇ alkyl: The term “C₁₋₇ alkyl” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a carbonatom of a hydrocarbon compound having from 1 to 7 carbon atoms, whichmay be aliphatic or alicyclic, and which may be saturated or unsaturated(e.g. partially unsaturated, fully unsaturated). Thus, the term “alkyl”includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussedbelow.

Examples of saturated alkyl groups include, but are not limited to,methyl (C₁), ethyl (C₂), propyl (C₃), butyl (C₄), pentyl (C₅), hexyl(C₆) and heptyl (C₇).

Examples of saturated linear alkyl groups include, but are not limitedto, methyl (C₁), ethyl (C₂), n-propyl (C₃), n-butyl (C₄), n-pentyl(amyl) (C₅), n-hexyl (C₆) and n-heptyl (C₇).

Examples of saturated branched alkyl groups include iso-propyl (C₃),iso-butyl (C₄), sec-butyl (C₄), tert-butyl (C₄), iso-pentyl (C₅), andneo-pentyl (C₅).

C₂₋₇ Alkenyl: The term “C₂₋₇ alkenyl” as used herein, pertains to analkyl group having one or more carbon-carbon double bonds.

Examples of unsaturated alkenyl groups include, but are not limited to,ethenyl (vinyl, —CH═CH₂), 1-propenyl (—CH═CH—CH₃), 2-propenyl (allyl,—CH—CH═CH₂), isopropenyl (1-methylvinyl, —C(CH₃)═CH₂), butenyl (C₄),pentenyl (C₅), and hexenyl (C₆).

C₂₋₇ alkynyl: The term “C₂₋₇ alkynyl” as used herein, pertains to analkyl group having one or more carbon-carbon triple bonds.

Examples of unsaturated alkynyl groups include, but are not limited to,ethynyl (ethinyl, —C≡CH) and 2-propynyl (propargyl, —CH₂—C≡CH).

C₃₋₇ cycloalkyl: The term “C₃₋₇ cycloalkyl” as used herein, pertains toan alkyl group which is also a cyclyl group; that is, a monovalentmoiety obtained by removing a hydrogen atom from an alicyclic ring atomof a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, thosederived from:

-   -   saturated monocyclic hydrocarbon compounds:        cyclopropane (C₃), cyclobutane (C₄), cyclopentane (C₅),        cyclohexane (C₆), cycloheptane (C₇), methylcyclopropane (C₄),        dimethylcyclopropane (C₅), methylcyclobutane (C₅),        dimethylcyclobutane (C₆), methylcyclopentane (C₆),        dimethylcyclopentane (C₇) and methylcyclohexane (C₇);    -   unsaturated monocyclic hydrocarbon compounds:        cyclopropene (C₃), cyclobutene (C₄), cyclopentene (C₅),        cyclohexene (C₆), methylcydopropene (C₄), dimethylcyclopropene        (C₅), methylcyclobutene (C₅), dimethylcyclobutene (C₆),        methylcyclopentene (C₆), dimethylcyclopentene (C₇) and        methylcyclohexene (C₇); and    -   saturated polycyclic hydrocarbon compounds:        norcarane (C₇), norpinane (C₇), norbornane (C₇).

C₃₋₂₀ heterocyclyl: The term “C₃₋₂₀ heterocyclyl” as used herein,pertains to a monovalent moiety obtained by removing a hydrogen atomfrom a ring atom of a heterocyclic compound, which moiety has from 3 to20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably,each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ringheteroatoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₃₋₇, C₅₋₆ etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆heterocyclyl”, as usedherein, pertains to a heterocyclyl group having 5 or 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limitedto, those derived from:

N₁: aziridine (C₃), azetidine (C₄), pyrrolidine (tetrahydropyrrole)(C₅), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C₅), 2H-pyrroleor 3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine (C₆),dihydropyridine (C₆), tetrahydropyridine (C₆), azepine (C₇);O₁: oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅), oxole(dihydrofuran) (C₅), oxane (tetrahydropyran) (C₆), dihydropyran (C₆),pyran (C₆), oxepin (C₇);S₁: thiirane (C₃), thietane (C₄), thiolane (tetrahydrothiophene) (C₅),thiane (tetrahydrothiopyran) (C₆), thiepane (C₇);O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇);O₃: trioxane (C₆);N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅), imidazoline(C₅), pyrazoline (dihydropyrazole) (C₅), piperazine (C₆);N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅), tetrahydroisoxazole(C₅), dihydroisoxazole (C₅), morpholine (C₆), tetrahydrooxazine (C₆),dihydrooxazine (C₆), oxazine (C₆);N₁S₁: thiazoline (C₅), thiazolidine (C₅), thiomorpholine (C₆);N₂O₁: oxadiazine (C₆);O₁S₁: oxathiole (C₅) and oxathiane (thioxane) (C₆); and,N₁O₁S₁: oxathiazine (C₆).

Examples of substituted monocyclic heterocyclyl groups include thosederived from saccharides, in cyclic form, for example, furanoses (C₅),such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse,and pyranoses (C₆), such as allopyranose, altropyranose, glucopyranose,mannopyranose, gulopyranose, idopyranose, galactopyranose, andtalopyranose.

C₅₋₂₀ aryl: The term “C₅₋₂₀ aryl”, as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from an aromaticring atom of an aromatic compound, which moiety has from 3 to 20 ringatoms. Preferably, each ring has from 5 to 7 ring atoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₅₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆ aryl” as used herein,pertains to an aryl group having 5 or 6 ring atoms.

The ring atoms may be all carbon atoms, as in “carboaryl groups”.

Examples of carboaryl groups include, but are not limited to, thosederived from benzene (i.e. phenyl) (C₆), naphthalene (C₁₀), azulene(C₁₀), anthracene (C₁₄), phenanthrene (C₁₄), naphthacene (C₁₈), andpyrene (C₁₆).

Examples of aryl groups which comprise fused rings, at least one ofwhich is an aromatic ring, include, but are not limited to, groupsderived from indane (e.g. 2,3-dihydro-1H-indene) (C₉), indene (C₉),isoindene (C₉), tetraline (1,2,3,4-tetrahydronaphthalene (C₁₀),acenaphthene (C₁₂), fluorene (C₁₃), phenalene (C₁₃), acephenanthrene(C₁₅), and aceanthrene (C₁₆).

Alternatively, the ring atoms may include one or more heteroatoms, as in“heteroaryl groups”. Examples of monocyclic heteroaryl groups include,but are not limited to, those derived from:

N₁: pyrrole (azole) (C₅), pyridine (azine) (C₆);O₁: furan (oxole) (C₅);S₁: thiophene (thiole) (C₅);N₁O₁: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);N₂O₁: oxadiazole (furazan) (C₅);N₃O₁: oxatriazole (C₅);N₁S₁: thiazole (C₅), isothiazole (C₅);N₂: imidazole (1,3-diazole) (C₅), pyrazole (1,2-diazole) (C₅),pyridazine (1,2-diazine) (C₆), pyrimidine (1,3-diazine) (C₆) (e.g.,cytosine, thymine, uracil), pyrazine (1,4-diazine) (C₆);N₃: triazole (C₅), triazine (C₆); and,N₄: tetrazole (C₅).

Examples of heteroaryl which comprise fused rings, include, but are notlimited to:

C₉ (with 2 fused rings) derived from benzofuran (O₁), isobenzofuran(O₁), indole (N₁), isoindole (N₁), indolizine (N₁), indoline (N₁),isoindoline (N₁), purine (N₄) (e.g., adenine, guanine), benzimidazole(N₂), indazole (N₂), benzoxazole (N₁O₁), benzisoxazole (N₁O₁),benzodioxole (O₂), benzofurazan (N₂O₁), benzotriazole (N₃),benzothiofuran (S₁), benzothiazole (N₁S₁), benzothiadiazole (N₂S);C₁₀ (with 2 fused rings) derived from chromene (O₁), isochromene (O₁),chroman (O₁), isochroman (O₁), benzodioxan (O₂), quinoline (N₁),isoquinoline (N₁), quinolizine (N₁), benzoxazine (N₁O₁), benzodiazine(N₂), pyridopyridine (N₂), quinoxaline (N₂), quinazoline (N₂), cinnoline(N₂), phthalazine (N₂), naphthyridine (N₂), pteridine (N₄);C₁₁ (with 2 fused rings) derived from benzodiazepine (N₂);C₁₃ (with 3 fused rings) derived from carbazole (N₁), dibenzofuran (O₁),dibenzothiophene (S₁), carboline (N₂), perimidine (N₂), pyridoindole(N₂); and,C₁₄ (with 3 fused rings) derived from acridine (N₁), xanthene (O₁),thioxanthene (S₁), oxanthrene (O₂), phenoxathiin (O₁S₁), phenazine (N₂),phenoxazine (N₁O₁), phenothiazine (N₁S₁), thianthrene (S₂),phenanthridine (N₁), phenanthroline (N₂), phenazine (N₂).

The above groups, whether alone or part of another substituent, maythemselves optionally be substituted with one or more groups selectedfrom themselves and the additional substituents listed below.

Halo: —F, —Cl, —Br, and —I.

Hydroxy: —OH.

Ether: —OR, wherein R is an ether substituent, for example, a C₁₋₇ alkylgroup (also referred to as a C₁₋₇ alkoxy group, discussed below), aC₃₋₂₀ heterocyclyl group (also referred to as a C₃₋₂₀ heterocyclyloxygroup), or a C₅₋₂₀ aryl group (also referred to as a C₅₋₂₀ aryloxygroup), preferably a C₁₋₇ alkyl group.

Alkoxy: —OR, wherein R is an alkyl group, for example, a C₁₋₇ alkylgroup. Examples of C₁₋₇ alkoxy groups include, but are not limited to,—OMe (methoxy), —OEt (ethoxy), —O(nPr) (n-propoxy), —O(iPr)(isopropoxy), —O(nBu) (n-butoxy), —O(sBu) (sec-butoxy), —O(iBu)(isobutoxy), and —O(tBu) (tert-butoxy).

Acetal: —CH(OR¹)(OR²), wherein R¹ and R² are independently acetalsubstituents, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocydylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group, or, in thecase of a “cyclic” acetal group, R¹ and R², taken together with the twooxygen atoms to which they are attached, and the carbon atoms to whichthey are attached, form a heterocyclic ring having from 4 to 8 ringatoms. Examples of acetal groups include, but are not limited to,—CH(OMe)₂, —CH(OEt)₂, and —CH(OMe)(OEt).

Hemiacetal: —CH(OH)(OR¹), wherein R¹ is a hemiacetal substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group.

Examples of hemiacetal groups include, but are not limited to,—CH(OH)(OMe) and —CH(OH)(OEt).

Ketal: —CR(OR¹)(OR²), where R¹ and R² are as defined for acetals, and Ris a ketal substituent other than hydrogen, for example, a C₁₋₇ alkylgroup, a C₃₋₂₀ heterocydyl group, or a C₅₋₂₀ aryl group, preferably aC₁₋₇ alkyl group. Examples ketal groups include, but are not limited to,—C(Me)(OMe)₂, —C(Me)(OEt)₂, —C(Me)(OMe)(OEt), —C(Et)(OMe)₂,—C(Et)(OEt)₂, and —C(Et)(OMe)(OEt).

Hemiketal: —CR(OH)(OR¹), where R¹ is as defined for hemiacetals, and Ris a hemiketal substituent other than hydrogen, for example, a C₁₋₇alkyl group, a C₃₋₂₀ heterocycyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of hemiacetal groups include,but are not limited to, —C(Me)(OH)(OMe), —C(Et)(OH)(OMe),—C(Me)(OH)(OEt), and —C(Et)(OH)(OEt).

Oxo (keto, -one): ═O.

Thione (thioketone): ═S.

Imino (imine): ═NR, wherein R is an imino substituent, for example,hydrogen, C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably hydrogen or a C₁₋₇ alkyl group. Examples of estergroups include, but are not limited to, ═NH, ═NMe, ═NEt, and ═NPh.

Formyl (carbaldehyde, carboxaldehyde): —C(═O)H.

Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example, aC₁₋₇ alkyl group (also referred to as C₁₋₇ alkylacyl or C₁₋₇ alkanoyl),a C₃₋₂₀ heterocyclyl group (also referred to as C₃₋₂₀ heterocyclylacyl),or a C₅₋₂₀ aryl group (also referred to as C₅₋₂₀ arylacyl), preferably aC₁₋₇ alkyl group. Examples of acyl groups include, but are not limitedto, —C(═O)CH₃ (acetyl), —C(═O)CH₂CH₃ (propionyl), —C(═O)C(CH₃)₃(t-butyryl), and —C(═O)Ph (benzoyl, phenone).

Carboxy (carboxylic acid): —C(═O)OH.

Thiocarboxy (thiocarboxylic acid): —C(═S)SH.

Thiolocarboxy (thiolocarboxylic acid): —C(═O)SH.

Thionocarboxy (thionocarboxylic acid): —C(═S)OH.

Imidic acid: —C(═NH)OH.

Hydroxamic acid: —C(═NOH)OH.

Ester (carboxylate, carboxylic acid ester, oxycarbonyl): —C(═O)OR,wherein R is an ester substituent, for example, a C₁₋₇ alkyl group, aC₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkylgroup. Examples of ester groups include, but are not limited to,—C(═O)OCH₃, —C(═O)OCH₂CH₃, —C(═O)OC(CH₃)₃, and —C(═O)OPh.

Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent,for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably a C₁₋₇ alkyl group. Examples of acyloxy groupsinclude, but are not limited to, —OC(═O)CH₃ (acetoxy), —OC(═O)CH₂CH₃,—OC(═O)C(CH₃)₃, —OC(═O)Ph, and —OC(═O)CH₂Ph.

Oxycarboyloxy: —OC(═O)OR, wherein R is an ester substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of ester groups include,but are not limited to, —OC(═O)OCH₃, —OC(═O)OCH₂CH₃, —OC(═O)OC(CH₃)₃,and —OC(═O)OPh.

Amino: —NR¹R², wherein R¹ and R² are independently amino substituents,for example, hydrogen, a C₁₋₇ alkyl group (also referred to as C₁₋₇alkylamino or di-C₁₋₇ alkylamino), a C₃₋₂₀ heterocyclyl group, or aC₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group, or, in the case ofa “cyclic” amino group, R¹ and R², taken together with the nitrogen atomto which they are attached, form a heterocyclic ring having from 4 to 8ring atoms. Amino groups may be primary (—NH₂), secondary (—NHR¹), ortertiary (—NHR¹R²), and in cationic form, may be quaternary (—⁺NR¹R²R³).Examples of amino groups include, but are not limited to, —NH₂, —NHCH₃,—NHC(CH₃)₂, —N(CH₃)₂, —N(CH₂CH₃)₂, and —NHPh. Examples of cyclic aminogroups include, but are not limited to, aziridino, azetidino,pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.

Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR¹R²,wherein R¹ and R² are independently amino substituents, as defined foramino groups. Examples of amido groups include, but are not limited to,—C(═O)NH₂, —C(═O)NHCH₃, —C(═O)N(CH₃)₂, —C(═O)NHCH₂CH₃, and—C(═O)N(CH₂CH₃)₂, as well as amido groups in which R¹ and R², togetherwith the nitrogen atom to which they are attached, form a heterocyclicstructure as in, for example, piperidinocarbonyl, morpholinocarbonyl,thiomorpholinocarbonyl, and piperazinocarbonyl.

Thioamido (thiocarbamyl): —C(═S)NR¹R², wherein R¹ and R² areindependently amino substituents, as defined for amino groups. Examplesof amido groups include, but are not limited to, —C(═S)NH₂, —C(═S)NHCH₃,—C(═S)N(CH₃)₂, and —C(═S)NHCH₂CH₃.

Acylamido (acylamino): —NR¹C(═O)R², wherein R¹ is an amide substituent,for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group,or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇, alkyl group, andR² is an acyl substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, preferably hydrogen or a C₁₋₇alkyl group. Examples of acylamide groups include, but are not limitedto, —NHC(═O)CH₃, —NHC(═O)CH₂CH₃, and —NHC(═O)Ph. R¹ and R² may togetherform a cyclic structure, as in, for example, succinimidyl, maleimidyl,and phthalimidyl:

Aminocarbonyloxy: —OC(═O)NR¹R², wherein R¹ and R² are independentlyamino substituents, as defined for amino groups. Examples ofaminocarbonyloxy groups include, but are not limited to, —OC(═O)NH₂,—OC(═O)NHMe, —OC(═O)NMe₂, and —OC(═O) NEt₂.

Ureido: —N(R¹)CONR²R³ wherein R² and R³ are independently aminosubstituents, as defined for amino groups, and R¹ is a ureidosubstituent, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇alkyl group. Examples of ureido groups include, but are not limited to,—NHCONH₂, —NHCONHMe, —NHCONHEt, —NHCONMe₂, —NHCONEt₂, —NMeCONH₂,—NMeCONHMe, —NMeCONHEt, —NMeCONMe₂, and —NMeCONEt₂.

Guanidino: —NH—C(═NH)NH₂.

Tetrazolyl: a five membered aromatic ring having four nitrogen atoms andone carbon atom,

Imino: ═NR, wherein R is an imino substituent, for example, for example,hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably H or a C₁₋₇alkyl group. Examples of imino groupsinclude, but are not limited to, ═NH, ═NMe, and ═NEt.

Amidine (amidino): —C(═NR)NR₂, wherein each R is an amidine substituent,for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group,or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group. Examples ofamidine groups include, but are not limited to, —C(═NH)NH₂, —C(═NH)NMe₂,and —C(═NMe)NMe₂.

Nitro: —NO₂.

Nitroso: —NO.

Azido: —N₃.

Cyano (nitrile, carbonitrile): —CN.

Isocyano: —NC.

Cyanato: —OCN.

Isocyanato: —NCO.

Thiocyano (thiocyanato): —SCN.

Isothiocyano (isothiocyanato): —NCS.

Sulfhydryl (thiol, mercapto): —SH.

Thioether (sulfide): —SR, wherein R is a thioether substituent, forexample, a C₁₋₇ alkyl group (also referred to as a C₁₋₇ alkylthiogroup), a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably aC₁₋₇ alkyl group. Examples of C₁₋₇ alkylthio groups include, but are notlimited to, —SCH₃ and —SCH₂CH₃.

Disulfide: —SS—R, wherein R is a disulfide substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group (also referred to herein as C₁₋₇ alkyldisulfide). Examples of C₁₋₇ alkyl disulfide groups include, but are notlimited to, —SSCH₃ and —SSCH₂CH₃.

Sulfine (sulfinyl, sulfoxide): —S(═O)R, wherein R is a sulfinesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfine groups include, but are not limited to, —S(═O)CH₃ and—S(═O)CH₂CH₃.

Sulfone (sulfonyl): —S(═O)₂R, wherein R is a sulfone substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group, including, for example, afluorinated or perfluorinated C₁₋₇ alkyl group. Examples of sulfonegroups include, but are not limited to, —S(═O)₂CH₃ (methanesulfonyl,mesyl), —S(═O)₂CF₃ (triflyl), —S(═O)₂CH₂CH₃ (esyl), —S(═O)₂C₄F₉(nonaflyl), —S(═O)₂CH₂CF₃ (tresyl), —S(═O)₂CH₂CH₂NH₂ (tauryl), —S(═O)₂Ph(phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl),4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl),4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsyl), and5-dimethylamino-naphthalen-1-ylsulfonate (dansyl).

Sulfinic acid (sulfino): —S(═O)OH, —SO₂H.

Sulfonic acid (sulfo): —S(═O)₂OH, —SO₃H.

Sulfinate (sulfinic acid ester): —S(═O)OR; wherein R is a sulfinatesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocycyl group,or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfinate groups include, but are not limited to, —S(═O)OCH₃(methoxysulfinyl; methyl sulfinate) and —S(═O)OCH₂CH₃ (ethoxysulfinyl;ethyl sulfinate).

Sulfonate (sulfonic acid ester): —S(═O)₂OR, wherein R is a sulfonatesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocycyl group,or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfonate groups include, but are not limited to, —S(═O)₂OCH₃(methoxysulfonyl; methyl sulfonate) and —S(═O)₂OCH₂CH₃ (ethoxysulfonyl;ethyl sulfonate).

Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxy substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of sulfinyloxy groupsinclude, but are not limited to, —OS(═O)CH₃ and —OS(═O)CH₂CH₃.

Sulfonyloxy: —OS(═O)₂R, wherein R is a sulfonyloxy substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of sulfonyloxy groupsinclude, but are not limited to, —OS(═O)₂CH₃ (mesylate) and—OS(═O)₂CH₂CH₃ (esylate).

Sulfate: —OS(═O)₂OR; wherein R is a sulfate substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfate groups include, butare not limited to, —OS(═O)₂OCH₃ and —SO(═O)₂OCH₂CH₃.

Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): —S(═O)NR¹R²,wherein R¹ and R² are independently amino substituents, as defined foramino groups. Examples of sulfamyl groups include, but are not limitedto, —S(═O)NH₂, —S(═O)NH(CH₃), —S(═O)N(CH₃)₂, —S(═O)NH(CH₂CH₃),—S(═O)N(CH₂CH₃)₂, and —S(═O)NHPh.

Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide):—S(═O)₂NR¹R², wherein R¹ and R² are independently amino substituents, asdefined for amino groups. Examples of sulfonamido groups include, butare not limited to, —S(═O)₂NH₂, —S(═O)₂NH(CH₃), —S(═O)₂N(CH₃)₂,—S(═O)₂NH(CH₂CH₃), —S(═O)₂N(CH₂CH₃)₂, and —S(═O)₂NHPh.

Sulfamino: —NR'S(═O)₂OH, wherein R¹ is an amino substituent, as definedfor amino groups. Examples of sulfamino groups include, but are notlimited to, —NHS(═O)₂OH and —N(CH₃)S(═O)₂OH.

Sulfonamino: —NR¹S(═O)₂R, wherein R¹ is an amino substituent, as definedfor amino groups, and R is a sulfonamino substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocycyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfonamino groups include,but are not limited to, —NHS(═O)₂CH₃ and —N(CH₃)S(═O)₂C₆H₅.

Sulfinamino: —NR₁S(═O)R, wherein R¹ is an amino substituent, as definedfor amino groups, and R is a sulfinamino substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfinamino groups include,but are not limited to, —NHS(═O)CH₃ and —N(CH₃)S(═O)C₆H₅.

Phosphino (phosphine): —PR₂, wherein R is a phosphino substituent, forexample, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group.Examples of phosphino groups include, but are not limited to, —PH₂,—P(CH₃)₂, —P(CH₂CH₃)₂, —P(t-Bu)₂, and —P(Ph)₂.

Phospho: —P(═O)₂.

Phosphinyl (phosphine oxide): —P(═O)R₂, wherein R is a phosphinylsubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group or a C₅₋₂₀aryl group. Examples of phosphinyl groups include, but are not limitedto, —P(═O)(CH₃)₂, —P(═O)(CH₂CH₃)₂, —P(═O)(t-Bu)₂, and —P(═O)(Ph)₂.

Phosphonic acid (phosphono): —P(═O)(OH)₂.

Phosphonate (phosphono ester): —P(═O)(OR)₂, where R is a phosphonatesubstituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or aC₅₋₂₀ aryl group. Examples of phosphonate groups include, but are notlimited to, —P(═O)(OCH₃)₂, —P(═O)(OCH₂CH₃)₂, —P(═O)(O-t-Bu)₂, and—P(═O)(OPh)₂.

Phosphoric acid (phosphonooxy): —OP(═O)(OH)₂.

Phosphate (phosphonooxy ester): —OP(═O)(OR)₂, where R is a phosphatesubstituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or aC₅₋₂₀ aryl group. Examples of phosphate groups include, but are notlimited to, —OP(═O)(OCH₃)₂, —OP(═O)(OCH₂CH₃)₂, —OP(═O)(O-t-Bu)₂, and—OP(═O)(OPh)₂.

Phosphorous acid: —OP(OH)₂.

Phosphite: —OP(OR)₂, where R is a phosphite substituent, for example,—H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group.Examples of phosphite groups include, but are not limited to,—OP(OCH₃)₂, —OP(OCH₂CH₃)₂, —OP(O-t-Bu)₂, and —OP(OPh)₂.

Phosphoramidite: —OP(OR¹)—NR²², where R¹ and R² are phosphoramiditesubstituents, for example, —H, a (optionally substituted) C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H,a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramiditegroups include, but are not limited to, —OP(OCH₂CH₃)—N(CH₃)₂,—OP(OCH₂CH₃)—N(i-Pr)₂, and —OP(OCH₂CH₂CN)—N(i-Pr)₂.

Phosphoramidate: —OP(═O)(OR¹)—NR²², where R¹ and R² are phosphoramidatesubstituents, for example, —H, a (optionally substituted) C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H,a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramidategroups include, but are not limited to, —OP(═O)(OCH₂CH₃)—N(CH₃)₂,—OP(═O)(OCH₂CH₃)—N(i-Pr)₂, and —OP(═O)(OCH₂CH₂CN)—N(i-Pr)₂.

Nitrogen Protecting Groups

Nitrogen protecting groups are well known in the art Preferred nitrogenprotecting groups are carbamate protecting groups that have the generalformula:

A large number of possible carbamate nitrogen protecting groups arelisted on pages 706 to 771 of Wuts, P. G. M. and Greene, T. W.,Protective Groups in Organic Synthesis, 4^(th) Edition,Wiley-Interscience, 2007, which is incorporated herein by reference.

Particularly preferred protecting groups include Alloc, Troc, Teoc, BOC,Doc, Hoc, TcBOC, Fmoc, 1-Adoc and 2-Adoc.

Hydroxyl Protecting Groups

Hydroxyl protecting groups are well known in the art. A large number ofsuitable groups are described on pages 16 to 366 of Wuts, P. G. M. andGreene, T. W., Protective Groups in Organic Synthesis, 4^(th) Edition,Wiley-Interscience, 2007, which is incorporated herein by reference.

Classes of particular interest include silyl ethers, methyl ethers,alkyl ethers, benzyl ethers, esters, benzoates, carbonates, andsulfonates.

Particularly preferred protecting groups include THP.

Proliferative Diseases One of ordinary skill in the art is readily ableto determine whether or not a candidate compound treats a proliferativecondition for any particular cell type. For example, assays which mayconveniently be used to assess the activity offered by a particularcompound are described in the examples below.

The term “proliferative disease” pertains to an unwanted or uncontrolledcellular proliferation of excessive or abnormal cells which isundesired, such as, neoplastic or hyperplastic growth, whether in vitroor in vivo.

Examples of proliferative conditions include, but are not limited to,benign, pre-malignant, and malignant cellular proliferation, includingbut not limited to, neoplasms and tumours (e.g. histocytoma, glioma,astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, bowel cancer, colon cancer, hepatoma, breastcancer, glioblastoma, cervical cancer, ovarian cancer, prostate cancer,testicular cancer, liver cancer, rectal cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, anal carcinoma, penile carcinoma, head and neck cancer,bladder cancer, pancreas cancer, brain cancer, sarcoma, osteosarcoma,Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases,fibroproliferative disorders (e.g. of connective tissues), andatherosclerosis. Cancers of particular interest include, but are notlimited to, leukemias and ovarian cancers.

Any type of cell may be treated, including but not limited to, lung,gastrointestinal (including, e.g. bowel, colon), breast (mammary),ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas,brain, and skin.

Cancers of particular interest include, but are not limited to, breastcancer (both ER positive and ER negative), pancreatic cancer, lungCancer and leukaemia.

Methods of Treatment

As described above, the present invention provides the use of a compoundof the first aspect of the invention in a method of therapy.

The term “therapeutically effective amount” is an amount sufficient toshow benefit to a patient Such benefit may be at least amelioration ofat least one symptom. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage, is within the responsibility of general practitioners and othermedical doctors.

A compound may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated. Examples of treatments and therapies include,but are not limited to, chemotherapy (the administration of activeagents, including, e.g. drugs); surgery; and radiation therapy.

Examples of chemotherapeutic agents include: erlotinib (TARCEVA®,Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU(fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®,Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin(cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin(CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology,Princeton, N.J.), trastuzumab (HERCEPTIN®, Genentech), temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide,CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen((2)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine,NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2,HPPD, and rapamycin.

More examples of chemotherapeutic agents include: oxaliplatin(ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent(SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin(folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib(TYKERB®, GSK572016, Glaxo Smith Kline), lonafamib (SARASAR™, SCH 66336,Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs),gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11,Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, II),vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478,AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib(GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa andcyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCBI-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlomaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.calicheamicin, calicheamicin gamma1I, calicheamicin omegaI1 (Angew Chem.Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin,marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide;edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche);ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylomithine (DMFO); retinoids such as retinoic acid; andpharmaceutically acceptable salts, acids and derivatives of any of theabove.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®;tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifinecitrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrolacetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole,RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX®(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipidkinase inhibitors; (vi) antisense oligonucleotides, particularly thosewhich inhibit expression of genes in signaling pathways implicated inaberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, suchas oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER² expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitorssuch as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptablesalts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” aretherapeutic antibodies such as alemtuzumab (Campath), bevacizumab(AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab(VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec),pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®,Genentech), tositumomab (Bexxar, Corixia), and the antibody drugconjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

Humanized monoclonal antibodies with therapeutic potential aschemotherapeutic agents in combination with the conjugates of theinvention include: alemtuzumab, apolizumab, aselizumab, atlizumab,bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumabmertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab,daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab,fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab,labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab,motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab,ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab,pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab,reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab,sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan,tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab,trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab,urtoxazumab, and visilizumab.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may comprise, in additionto the active ingredient, i.e. a compound of formula I, apharmaceutically acceptable excipient, carrier, buffer, stabiliser orother materials well known to those skilled in the art. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive ingredient. The precise nature of the carrier or other materialwill depend on the route of administration, which may be oral, or byinjection, e.g. cutaneous, subcutaneous, or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carrier oran adjuvant. Liquid pharmaceutical compositions generally comprise aliquid carrier such as water, petroleum, animal or vegetable oils,mineral oil or synthetic oil. Physiological saline solution, dextrose orother saccharide solution or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol may be included. A capsule may comprise asolid carrier such a gelatin.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Dosage

It will be appreciated by one of skill in the art that appropriatedosages of the compound can vary from patient to patient. Determiningthe optimal dosage will generally involve the balancing of the level oftherapeutic benefit against any risk or deleterious side effects. Theselected dosage level will depend on a variety of factors including, butnot limited to, the activity of the particular compound, the route ofadministration, the time of administration, the rate of excretion of thecompound, the duration of the treatment, other drugs, compounds, and/ormaterials used in combination, the severity of the condition, and thespecies, sex, age, weight, condition, general health, and prior medicalhistory of the patient. The amount of compound and route ofadministration will ultimately be at the discretion of the physician,veterinarian, or clinician, although generally the dosage will beselected to achieve local concentrations at the site of action whichachieve the desired effect without causing substantial harmful ordeleterious side-effects.

Administration can be effected in one dose, continuously orintermittently (e.g., in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell(s) being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician, veterinarian, or clinician.

In general, a suitable dose of the active compound is in the range ofabout 100 ng to about 25 mg (more typically about 1 μg to about 10 mg)per kilogram body weight of the subject per day. Where the activecompound is a salt, an ester, an amide, a prodrug, or the like, theamount administered is calculated on the basis of the parent compoundand so the actual weight to be used is increased proportionately.

In one embodiment, the active compound is administered to a humanpatient according to the following dosage regime: about 100 mg, 3 timesdaily.

In one embodiment, the active compound is administered to a humanpatient according to the following dosage regime: about 150 mg, 2 timesdaily.

In one embodiment, the active compound is administered to a humanpatient according to the following dosage regime: about 200 mg, 2 timesdaily.

For the prevention or treatment of disease, the appropriate dosage ofthe compound of the invention will depend on the type of disease to betreated, as defined above, the severity and course of the disease,whether the molecule is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the antibody, and the discretion of the attending physician. Themolecule is suitably administered to the patient at one time or over aseries of treatments. Depending on the type and severity of the disease,about 1 μg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of molecule is an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. An exemplarydosage of compound to be administered to a patient is in the range ofabout 0.1 to about 10 mg/kg of patient weight. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. An exemplary dosing regimen comprises a course ofadministering an initial loading dose of about 4 mg/kg, followed byadditional doses every week, two weeks, or three weeks of a compound.Other dosage regimens may be useful. The progress of this therapy iseasily monitored by conventional techniques and assays.

Includes Other Forms

Unless otherwise specified, included in the above are the well knownionic, salt, solvate, and protected forms of these substituents. Forexample, a reference to carboxylic acid (—COOH) also includes theanionic (carboxylate) form (—COO⁻), a salt or solvate thereof, as wellas conventional protected forms. Similarly, a reference to an aminogroup includes the protonated form (—N⁺HR¹R²), a salt or solvate of theamino group, for example, a hydrochloride salt, as well as conventionalprotected forms of an amino group. Similarly, a reference to a hydroxylgroup also includes the anionic form (—O⁻), a salt or solvate thereof,as well as conventional protected forms.

Isomers, Salts and Solvates

Certain compounds may exist in one or more particular geometric,optical, enantiomeric, diasteriomeric, epimeric, atropic,stereoisomeric, tautomeric, conformational, or anomeric forms, includingbut not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, andr-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d-and l-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn-and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axialand equatorial forms; boat-, chair-, twist-, envelope-, andhalfchair-forms; and combinations thereof, hereinafter collectivelyreferred to as “isomers” (or “isomeric forms”).

Preferably compounds of the present invention have the followingstereochemistry at the C₁₁ position:

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers”, as used herein, are structural (orconstitutional) isomers (i.e. isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH₂OH.Similarly, a reference to ortho-chlorophenyl is not to be construed as areference to its structural isomer, meta-chlorophenyl. However, areference to a class of structures may well include structurallyisomeric forms falling within that class (e.g. C₁₋₇ alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl;methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol (illustrated below), imine/enamine,amide/imino alcohol, amidine/amidine, nitroso/oxime,thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including (wholly or partially)racemic and other mixtures thereof. Methods for the preparation (e.g.asymmetric synthesis) and separation (e.g. fractional crystallisationand chromatographic means) of such isomeric forms are either known inthe art or are readily obtained by adapting the methods taught herein,or known methods, in a known manner.

Unless otherwise specified, a reference to a particular compound alsoincludes ionic, salt, solvate, and protected forms of thereof, forexample, as discussed below.

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66,1-19 (1977).

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g. —COOH may be —COO), then a salt may be formed witha suitable cation.

Examples of suitable inorganic cations include, but are not limited to,alkali metal ions such as Na⁺ and K⁺, alkaline earth cations such asCa²⁺ and Mg²⁺, and other cations such as Al³⁺. Examples of suitableorganic cations include, but are not limited to, ammonium ion (i.e. NH₄⁺) and substituted ammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺).Examples of some suitable substituted ammonium ions are those derivedfrom: ethylamine, diethylamine, dicyclohexylamine, triethylamine,butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine,benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, aswell as amino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may becationic (e.g. —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to,those derived from the following organic acids: 2-acetyoxybenzoic,acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric,edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic,gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic,methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic,phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic,succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examplesof suitable polymeric organic anions include, but are not limited to,those derived from the following polymeric acids: tannic acid,carboxymethyl cellulose.

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the active compound. The term “solvate” is usedherein in the conventional sense to refer to a complex of solute (e.g.active compound, salt of active compound) and solvent. If the solvent iswater, the solvate may be conveniently referred to as a hydrate, forexample, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

Compounds of formula I include compounds where a nucleophilic solvent(H₂O, R^(A)OH, R^(A)NH₂, R^(A)SH) adds across the imine bond of the PBDmoiety, which is illustrated below where the solvent is water or analcohol (R^(A)OH, where R^(A) is an ether substituent as describedabove):

These forms can be called the carbinolamine and carbinolamine etherforms of the PBD. The balance of these equilibria depend on theconditions in which the compounds are found, as well as the nature ofthe moiety itself.

These compounds may be isolated in solid form, for example, bylyophilisation.

General Synthetic Routes

Compounds of formula I where R¹⁰ and R¹¹ together form a double bond maybe synthesised from compounds of formula 2:

R′¹⁰ is a nitrogen protecting group and R′¹¹ is O—R¹², wherein R¹² is Hor a hydroxyl protecting group. Such techniques are well known in theart, and are described, for example, in Wuts, P. G. M. and Greene, T.W., Protective Groups in Organic Synthesis, 4^(th) Edition,Wiley-Interscience, 2007. If both nitrogen and hydroxyl protectinggroups are present, these are preferably selected to be removable by thesame conditions.

If this deprotection is carried out in a solvent of formula HQR^(Q),then R¹⁰ and R¹¹ will be H and QR^(Q) respectively. Alternatively, thesegroups may be introduced by adding the compound to a different solventto that in which the deprotection is carried out.

The conversion of compounds of formula I as discussed above to thosehaving R¹¹ as SO_(x)M may be achieved by the addition of the appropriatebisulphite salt or sulphinate salt, followed by a purification step.Further methods are described in GB 2 053 894, which is hereinincorporated by reference.

Compounds of formula 2 can be made by the coupling of compounds ofFormula 3 and Formula 4:

under standard amide bond formation conditions, e.g. in the presence ofHOBt or DMAP and EDCl.

Compounds of formula 3 can be synthesised from compounds of formula 5:

where R′⁸ is a C₁₄ alkyl group, e.g. methyl. This deprotection of thecarboxyl group may be carried out using standard means, e.g. treatmentwith base.

Compounds of formula 5 can be synthesised in general following themethods described in WO 00/12506 and WO 2007/039752, which are hereinincorporated by reference. In particular, the butanoic acid side chaincan be introduced at any stage in the synthesis, usually withappropriate protecting groups in place. For example, the side chain canbe formed by coupling a protected or precursor form to a hydroxy groupon the bezene ring using e.g. Mitsunobo coupling.

Compounds of formula 4 can be synthesised using the methods disclosed inWO 2005/085177, which are incorporated herein by reference. Reference isalso made to the disclosure of WO 2007/039752.

DNA Binding The ability of the compounds to bind to DNA, and inparticular oligonucleotides, can be measured using an Ion PairReversed-Phase HPLC assay, as described in Rahman, K. M., et al.,Journal of the American Chemical Society 2009, 131, 13756 andNarayanaswamy, M., et al., Analytical Biochemistry 2008, 374, 173. TheDNA binding affinity can also be evaluated by using a calf-thymus DNAthermal denaturation assay, as described in Wells, G., et al., Journalof Medicinal Chemistry 2006, 49, 5442; Jenkins, T. C., et al., Journalof Medicinal Chemistry 1994, 37, 4529; and Gregson, S. J., et al.,Journal of Medicinal Chemistry 2001, 44, 737.

Further Preferences C₂

It may be preferred in any of the embodiments that the C₂ carbon is asp² centre, so that when R² is selected from any of the followinggroups:

—H, —OH, —CN, —R, —OR, halo, —O—SO₂—R, —CO₂R and —CORthere is a double bond between C2 and C3.

When R² is selected from any of the following groups:

═O, ═CH₂, ═CHR, ═CHRR′

there cannot be a double bond between C2 and C3.

In further embodiments, there is no double bond between C2 and C3, andR² is H.

R²

R² is selected from —H, —OH, ═O, ═CH₂, —CN, —R, OR, halo, dihalo, ═CHR,═CHRR′, —O—SO₂—R, CO₂R and COR.

In some embodiments, R² may be selected from —H, —OH, ═O, ═CH₂, —CN, —R,—OR, ═CHR, ═CRR′, —O—SO₂—R, —CO₂R and —COR.

In some embodiments, R² may be selected from —H, ═CH₂, —R, ═CHR, and═CRR′.

In one embodiment, R² is H.

In one embodiment, R² is ═O.

In one embodiment, R² is ═CH₂.

In one embodiment, R² is ═CHR. Within the PBD compound, the group ═CHRmay have either configuration shown below:

In one embodiment, the configuration is configuration (C1).

In one embodiment, R² is ═CRR′.

In one embodiment, R² is ═CF₂.

In one embodiment, R² is R.

In one embodiment, R² is optionally substituted C₅₋₂₀ aryl.

When R² is optionally substituted C₅₋₂₀ aryl, it may preferably beoptionally substituted C₅₋₇ aryl or C₈₋₁₀ aryl. R² may furtherpreferably be optionally substituted phenyl, optionally substitutednapthyl, optionally substituted pyridyl, optionally substitutedquinolinyl or isoquinolinyl. Of these groups, optionally substitutedphenyl is most preferred.

When R² is optionally substituted C₅₋₂₀ aryl, it may preferably bear oneto three substituent groups, with 1 and 2 being more preferred, andsingly substituted groups being most preferred. The substituents may beany position.

Where R² is a C₅₋₇ aryl group, a single substituent is preferably on aring atom that is not adjacent the bond to the remainder of thecompound, i.e. it is preferably 1 or y to the bond to the remainder ofthe compound. Therefore, where the C₅₋₇ aryl group is phenyl, thesubstituent is preferably in the meta- or para-positions, and morepreferably is in the para-position.

In one embodiment, R² is selected from:

-   -   where the asterisk indicates the point of attachment.

Where R² is a C₈₋₁₀ aryl group, for example quinolinyl or isoquinolinyl,it may bear any number of substituents at any position of the quinolineor isoquinoline rings. In some embodiments, it bears one, two or threesubstituents, and these may be on either the proximal and distal ringsor both (if more than one substituent).

When R² is optionally substituted C₅₋₂₀ aryl, the substituents may beselected from: halo, hydroxyl, ether, formyl, acyl, carboxy, ester,acyloxy, amino, amido, acylamido, aminocarbonyloxy, ureido, nitro, cyanoand thioether.

When R² is optionally substituted C₅₋₂₀ aryl, the substituents may beselected from the group consisting of R, OR, SR, NRR′, NO₂, halo, CO₂R,COR, CONH₂, CONHR, and CONRR′.

If a substituent on R² is halo, it is preferably F or CI, morepreferably CI.

If a substituent on R² is ether, it may in some embodiments be an alkoxygroup, for example, a C₁₋₇ alkoxy group (e.g. methoxy, ethoxy) or it mayin some embodiments be a C₅₋₇ aryloxy group (e.g. phenoxy, pyridyloxy,furanyloxy).

If a substituent on R² is C₁₋₇ alkyl, it may preferably be a C₁₋₄ alkylgroup (e.g. methyl, ethyl, propyl, butyl).

If a substituent on R² is C₃₋₇ heterocyclyl, it may in some embodimentsbe C₆ nitrogen containing heterocyclyl group, e.g. morpholino,thiomorpholino, piperidinyl, piperazinyl. These groups may be bound tothe rest of the PBD moiety via the nitrogen atom. These groups may befurther substituted, for example, by C₁₋₄ alkyl groups.

If a substituent on R² is bis-oxy-C₁₋₃ alkylene, this is preferablybis-oxy-methylene or bis-oxy-ethylene.

Particularly preferred substituents for R² include methoxy, ethoxy,fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholinoand methyl-thienyl.

Particularly preferred substituted R² groups include, but are notlimited to, 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl,3-ethoxy-phenyl, 4-fluoro-phenyl, 4-chloro-phenyl,3,4-bisoxymethylene-phenyl, 4-methylthienyl, 4-cyanophenyl,4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl andisoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and naphthyl.

In one embodiment, R² is optionally substituted C₁₋₁₂ alkyl.

When R² is optionally substituted C₁₋₁₂ alkyl, it may be selected from:

(a) C₁₋₅ saturated aliphatic alkyl;(b) C₃₋₆ saturated cycloalkyl;(c)

wherein each of R²¹, R²² and R²³ are independently selected from H, C₁₋₃saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where thetotal number of carbon atoms in the R¹² group is no more than 5;(d)

wherein one of R^(25a) and R^(25b) is H and the other is selected from:phenyl, which phenyl is optionally substituted by a group selected fromhalo methyl, methoxy; pyridyl; and thiophenyl; and(e)

where R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted bya group selected from halo methyl, methoxy; pyridyl; and thiophenyl.

When R² is C₁₋₅ saturated aliphatic alkyl, it may be methyl, ethyl,propyl, butyl or pentyl.

In some embodiments, it may be methyl, ethyl or propyl (n-pentyl orisopropyl). In some of these embodiments, it may be methyl. In otherembodiments, it may be butyl or pentyl, which may be linear or branched.

When R² is C₃₋₆ saturated cycloalkyl, it may be cyclopropyl, cyclobutyl,cyclopentyl or cyclohexyl. In some embodiments, it may be cyclopropyl.

When R² is

each of R²¹, R²² and R²³ are independently selected from H, C₁₋₃saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where thetotal number of carbon atoms in the R² group is no more than 5. In someembodiments, the total number of carbon atoms in the R² group is no morethan 4 or no more than 3.

In some embodiments, one of R²¹, R²² and R²³ is H, with the other twogroups being selected from H, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃alkynyl and cyclopropyl.

In other embodiments, two of R²¹, R²² and R²³ are H, with the othergroup being selected from H, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃alkynyl and cyclopropyl.

In some embodiments, the groups that are not H are selected from methyland ethyl. In some of these embodiments, the groups that are not H aremethyl.

In some embodiments, R²¹ is H.

In some embodiments, R²² is H.

In some embodiments, R²³ is H.

In some embodiments, R²¹ and R²² are H.

In some embodiments, R²¹ and R²³ are H.

In some embodiments, R²² and R²³ are H.

When R² is

one of R^(25a) and R^(25b) is H and the other is selected from: phenyl,which phenyl is optionally substituted by a group selected from halo,methyl, methoxy; pyridyl; and thiophenyl. In some embodiments, the groupwhich is not H is optionally substituted phenyl. If the phenyl optionalsubstituent is halo, it is preferably fluoro. In some embodiment, thephenyl group is unsubstituted.

When R² is

R²⁴, R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted bya group selected from halo methyl, methoxy; pyridyl; and thiophenyl. Ifthe phenyl optional substituent is halo, it is preferably fluoro. Insome embodiment, the phenyl group is unsubstituted. In some embodiments,R²⁴ is selected from H, methyl, ethyl, ethenyl and ethynyl. In some ofthese embodiments, R²⁴ is selected from H and methyl.

In one embodiment, R² is halo or dihalo. In one embodiment, R² is —F or—F₂, which substituents are illustrated below as (C3) and (C4)respectively:

R² may preferably selected from ═CH₂, ═CH—R, where R is more preferablyan optionally substituted C₁₋₄ alkyl group, and —R, where R is morepreferably an optionally substituted C₅₋₂₀ aryl group. Particularlypreferred groups for R² include ═CH₂, ═CH-Me, and an optionallysubstituted phenyl group.

R⁷

R⁷ is selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′, nitro, Me₃Snand halo;

R⁷ may preferably be selected from H, OR, SH, SR, NH₂, NHR, NRR′, andhalo.

R⁷ may more preferably be selected from H and OR.

In some embodiments, R⁷ is OR, and more particularly OR^(7A), whereR^(7A) is independently optionally substituted C₁₋₇ alkyl.

R^(7A) may be selected from optionally substituted saturated C₁₋₇ alkyland optionally substituted C₂₋₄ alkenyl.

R^(7A) may preferably be selected from Me, CH₂Ph and allyl.

R¹⁰/R¹¹

R¹⁰ and R¹¹ either together form a double bond, or are selected from Hand QR^(Q) respectively, where Q is selected from O, S and NH and R^(Q)is H or C₁₋₇ alkyl or H and SO_(x)M, where x is 2 or 3, and M is amonovalent pharmaceutically acceptable cation;

In some embodiments, R¹⁰ and R¹¹ form a double bond together.

In some embodiments, R¹⁰ is H and R¹¹ is OR^(Q). In these embodiments,R^(Q) may preferably be selected from H or Me.

In some embodiments, R¹⁰ is H and R¹¹ is SO_(x)M. x may preferably be 3,and M may preferably be Na⁺.

R¹

-   -   R¹ is C₁-4 alkyl.

R¹ may preferably be C₁₋₂ alkyl, and more preferably methyl.

A

A is either:

where X and Y are selected from: CH and NMe; COH and NMe; CH and S; Nand NMe;

N and S.

In some embodiments, A is A1.

In some embodiments, A is A2.

In any of these embodiments, X and Y may preferably be selected from CHand NMe; CH and S; N and NMe; and N and S. X and Y may more preferablybe selected from CH and NMe; and N and NMe.

In some embodiments X and Y are CH and NMe.

In some embodiments X and Y are N and NMe.

B

B is either a single bond or

where X and Y are as defined above, but independently selected.

In some embodiments, B is a single bond.

In some embodiments, B is B1.

In any of these embodiments, X and Y may preferably be selected from CHand NMe; CH and S; N and NMe; and N and S. X and Y may more preferablybe selected from CH and NMe; and N and NMe.

In some embodiments X and Y are CH and NMe.

In some embodiments X and Y are N and NMe.

FIGURES

FIGS. 1A and 1B shows the results of an assay to determine the maximumtolerated dose of two compounds of the invention;

FIG. 2 shows the result of an assay to determine the in vivo activity ofa compound of the invention; and

FIG. 3 shows the result of another assay to determine the in vivoactivity of the same compound as in FIG. 2.

EXAMPLES General methods

Optical rotations were measured on an ADP 220 polarimeter (BellinghamStanley Ltd) and concentrations (c) are given in g/100 mL. Meltingpoints were measured using a digital melting point apparatus(Electrothermal). IR spectra were recorded on a Perkin-Elmer Spectrum1000 FT IR Spectrometer. ¹H and ¹³C NMR spectra were acquired at 300 Kusing a Bruker Advance NMR spectrometer at 400 and 100 MHz,respectively. Chemical shifts are reported relative to TMS (δ=0.0 ppm),and signals are designated as s (singlet), d (doublet), t (triplet), dt(double triplet), dd (doublet of doublets), ddd (double doublet ofdoublets) or m (multiplet), with coupling constants given in Hertz (Hz).A pro-PBD numbering system is used for carbon and proton assignments forsynthetic intermediates (i.e., based on the final tricyclic PBD ringsystem). Mass spectrometry data were collected using a Waters MicromassZQ instrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA.Waters Micromass ZQ parameters used were: Capillary (kV), 3.38; Cone(V), 35; Extractor (V), 3.0; Source temperature (° C.), 100; DesolvationTemperature (° C.), 200; Cone flow rate (L/h), 50; De-solvation flowrate (L/h), 250. High-resolution mass spectrometry data were recorded ona Waters Micromass QTOF Global in positive W-mode using metal-coatedborosilicate glass tips to introduce the samples into the instrument.Thin Layer Chromatography (TLC) was performed on silica gel aluminumplates (Merck 60, F₂₅₄), and flash chromatography utilized silica gel(Merck 60, 230-400 mesh ASTM). Parallel reactions were carried out usinga Radleys™ Green House Synthesizer and parallel purifications werecarried out using an IST Vacmaster™. For reactions carried out inparallel, solvents were evaporated using a Genevac VC 2000D (GenevacTechnologies, UK). Purified compounds were freeze dried using aHeto-Lyolab 3000 freeze drier. Hydrogenation reactions were carriedusing UHP-60H hydrogen generator attached to a Parr hydrogenationapparatus. Synthetic building blocks were purchased from MaybridgeChemicals (UK), Bachem Chemicals (USA) and Sigma-Aldrich (UK). Reagentsand solvents were purchased from Sigma-Aldrich (UK).

Synthesis of Key Intermediates (a) Methyl4-(4-(tert-butoxycarbonylamino)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(5)

(i) 2-(trichloroacetyl)-1-methyl pyrrole (2)

A solution of N-methyl pyrrole (1) (113.06 g, 1.39 mol, 1.0 eq) in dryether (350 mL) was added drop wise over a period of 1 hour and 10minutes to a stirred solution of trichloroacetyl chloride (254 g, 1.39mol, 1.0 eq) in dry ether (350 mL) in a 2 L, 3 necked flask. HCl gasproduced in the reaction was removed by flushing with nitrogen. Thereaction mixture was allowed to stir for 1.5 hours and progress of thereaction was monitored regularly by TLC and LCMS. After 1.5 hours thereaction was quenched using 1 M K₂CO₃ solution. The reaction mixture wasextracted with ethyl acetate (3×) and the organic layers were combinedand concentrated in vacuo. The crystalline residue was washed withn-hexane and finally dried under vacuum. Yield—281.18 g, 79.5%, NMRcompared with literature

IR, (FTIR, v_(max)/cm⁻¹) 3299, 3121, 3008, 2954, 1789, 1674, 1521, 1406,1206, 1100, 980, 881, 757; ¹H-NMR (CDCl₃, 400 MHz) δ 7.42 (1H, dd,J=4.4, 1.6 Hz), 6.97 (1H, t, J=1.6 Hz), 6.22 (1H, dd, J=4.4, 2.4 Hz)3.97 (3H, s); ¹³C NMR (CDCl₃, 400 MHz): δ 133.6, 124.0, 122.4, 108.9,38.5.

(ii) 1-(4-bromo-1-methyl-1H-pyrrol-2-yl)-2,2,2-trichloroethanone (3)

NBS (N-Bromosuccinimide, 2.36 g, 13.24 mmol, 1.0 equiv.) was added to astirred solution of 2-(trichloroacetyl)-1-methylpyrrole (2) (3 g, 13.24mmol, 1.0 equiv.) in anhydrous THF (35 mL) at −10° C. The reactionmixture was kept at −10° C. for 2 hours and then left to reach roomtemperature (ca. 4 hours). Excess THF was evaporated in vacuum and thesolid was re-dissolved in a mixture of EtOAc/n-hexane (1:9). Theresulting mixture was filtered through a plug of silica, and thefiltrate was evaporated in vacuo. The resulting solid was recrystallisedfrom n-hexane to give 3 (3.55 g, 88%). IR (FTIR, v_(max)/cm⁻¹): 3148,2956, 1669 (C═O), 1458, 1215, 1189, 1062, 923, 842, 823, 748, 678;¹H-NMR (CDCl₃, 400 MHz) δ 7.46 (1H, d, J=2.0 Hz), 6.95 (1H, d, J=1.6 Hz)3.95 (3H, s); ¹³C NMR (CDCl₃, 100 MHz): δ 172.4, 132.8, 124.6, 132.2,96.1, 38.7; EIMS m/z (+EI) calc. for C₇HsBrCl₃NO (M)⁺ 305.38, LCMSanalysis found 306.86 (M+H)⁺

(iii) Methyl 4-bromo-1-methyl-1H-pyrrole-2-carboxylate (4)

To a stirred solution of1-(4-bromo-1-methyl-1H-pyrrol-2-yl)-2,2,2-trichloro-ethanone (3)(3.28 g,10.74 mmol, 1 eq.) in dry MeOH (30 mL), a solution of sodium methoxide(0.5 mL) was added by a syringe. The sodium methoxide solution wasprepared from NaH 60% in mineral oil (43 mg, 1.07 mmol, 0.1 eq.), whichwas previously washed with n-hexane. The solution was heated to refluxover a period of 30 minutes, when the TLC analysis showed completeconsumption of the starting material. A few drops of concentrated H₂SO₄were added to the solution to neutralise the base (pH 2). The excessMeOH was evaporated in vacuum and the resulting oil was redissolved inEtOAc (50 mL) and washed with water (40 mL). The aqueous layer wasextracted with EtOAc (3×40 mL), and the organic phases were combined,dried (MgSO₄), filtered and concentrated in vacuum to afford the productas a pale white solid. (2.28 g, 97%). IR (FTIR, v_(max)/cm⁻¹): 3138,2948, 1692, 1472, 1334, 1245, 1115, 1082, 921, 823, 753; ¹H-NMR (400MHz, CDCl₃): δ 6.89 (d, 1H, J=2.0 Hz), 6.76 (d, 1H, J=2.0 Hz), 3.89 (s,3H), 3.81 (s, 3H); ¹³C-NMR (100 MHz, CDCl₃): δ 160.8, 128.7, 122.9,119.2, 95.1, 51.2, 36.9; EIMS m/z (+EI) calc. for C₇H₈BrNO₂ (M)⁺ 218.05found 219.26 (M+H)⁺

(iv) Methyl4-(4-(tert-butoxycarbonylamino)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(5)

A catalytic amount of tetrakis(triphenylphosphine)palladium, Pd(PPh₃)₄(0.477 g, 0.413, 0.06 eq) was added to a solution of 4 (1.5 g, 6.88mmol, 1 eq) and (4-((tert-butoxycarbonyl)amino)phenyl)boronic acid (1.57g, 6.88 mmol, 1.20 eq) in a 9:3:1 combination (13.5 ml) of EtOH, tolueneand water in the presence of K₂CO₃ (2.856 g, 3 eq.) in a 10-20 mLmicrowave vial containing a magnetic stirrer. The reaction vessel wasflushed with nitrogen during each addition. The reaction mixture wassealed in an inert N₂ atmosphere and heated with microwave radiation inan EMRYS™ Optimizer Microwave Station (Personal Chemistry) at 100° C.for 12 minutes. After LCMS and TLC analysis revealed completion of thereaction, the cooled reaction mixture was diluted with water (50 mL),extracted with EtOAc (3×40 mL), the filtrates combined, dried over MgSO₄and concentrated under vacuum. The resulting oil was subjected to flashchromatography (n-hexane/EtOAc 9:1) to give 5 (Yield—2.2 g, 97%). IR(FTIR, v^(max)/cm⁻): 3353, 2975, 1696, 1521, 1441, 1366, 1264, 1235,1209, 1058, 822, 799, 657; ¹H-NMR (400 MHz, CDCl₃): δ 7.40 (d, 2H, J=8.8Hz), 7.33 (d, 2H, J=8.8 Hz), 7.16 (d, 1H, J=2.0 Hz), 7.02 (d, 1H,J=2.0), 6.45 (br s, 1H), 3.95 (s, 3H), 3.83 (s, 3H), 1.52 (s, 9H);¹³C-NMR (100 MHz, CDCl₃): δ 161.7, 152.8, 136.5, 129.5, 125.9, 125.6,123.7, 123.0, 119.0, 114.6, 80.5, 51.1, 36.9, 28.4; EIMS m/z (+EI) calc.for C₁₈H₂₂N₂O₄ (M)⁺ 330.38 found 330.46 (M+H)⁺

(b)4-(4-(tert-butoxycarbonylamino)phenyl)-1-methyl-1H-pyrrole-2-carboxylicacid (6)

A 0.5 M solution of NaOH (2.0 eq) was added to a solution of 5 (1.0 g,3.027 mmol) in dioxane (40 mL). The reaction mixture was allowed to stirat room temperature for 6 hours at which point TLC revealed completionof reaction. Excess 1,4-dioxane was evaporated under vacuum and theresidue was diluted with water. The resulting solution was acidifiedwith 0.5 M HCl. The product was extracted from water with 2× ethylacetate (100 mL×2) and the organic layers were combined, washed withbrine, dried over MgSO₄ and concentrated in vacuo. The product waspurified using flash chromatography (ethyl acetate/n-hexane 2:8).Yield—0.92 g, 96.8%. IR (FTIR, v^(max)/cm⁻¹): 3371, 2979, 1698, 1671,1522, 1445, 1367, 1285, 1161, 1112, 1047, 823, 803, 762, 714, 631;¹H-NMR (400 MHz, CDC₃): δ 8.33 (1H, s), 7.55 (d, 2H, J=8.8 Hz), 7.50 (d,2H, J=8.8 Hz), 7.36 (d, 1H, J=2.0 Hz), 7.22 (d, 1H, J=2.0), 3.97 (s,3H), 1.50 (s, 9H); ¹³C-NMR (100 MHz, CDCl₃): δ 162.3, 153.7, 138.6,123.0, 127.1, 126.0, 124.4, 124.0, 119.5, 115.1, 79.9, 36.9, 28.6; EIMSm/z (+EI) calc. for C₁₇H₂₀N₂O₄ (M)⁺ 316.35 found 315.16 (M+H)⁺

(c)

(i) Methyl 4-(4-aminophenyl)-1-methyl-1H-pyrrole-2-carboxylate (7)

5 (1 g, 3.027 mmol) was dissolved in a small volume of MeOH and 4M HClin dioxane (15 mL) was added slowly to the stirring solution. Thereaction mixture was stirred for 6 hours at which point TLC showedcompletion of reaction. Excess solvent was evaporated under vacuum toobtain a brown coloured solid 7. The solid product was subjected toflash chromatography (n-hexane/EtOAc 9:1) to give pure 7 (065 g, 94.2%).IR (FTIR, v^(max)/cm⁻¹): 3366, 2987, 1688, 1629, 1566, 1422, 1372, 1262,1181, 1103, 1067, 951, 821, 784, 756; ¹H-NMR (400 MHz, CDCl₃): δ 7.28(2H, d, J=8.4 Hz), 7.11 (1H, d, J=2.0 Hz), 6.96 (1H, d, J=2.0 Hz), 6.68(d, 2H, J=8.0 Hz), 3.94 (s, 3H), 3.83 (s, 3H); ¹³C-NMR (100 MHz, CDCl₃):δ 161.7, 144.7, 126.2, 125.4, 125.2, 115.5, 114.4, 51.0, 36.8; EIMS m/z(+EI) calc. for C₁₃H₁₄N₂O₂ (M)⁺ 230.26 found 231.1 (M+H)⁺

(ii) Methyl4-(4-(4-(4-aminophenyl)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(8)

0.2 g boc protected 6 (0.63 mmol, 1.2 eq) was dissolved in DMF (5 mL) towhich 2.0 eq of EDCl and 2.5 eq of DMAP were added and the mixture wasallowed to stir for 30 minutes. At this point methyl4-amino-1-methyl-1H-pyrrole-2-carboxylate (80.9 mg, 0.52 mmol, 1.0 eq)was added and the reaction mixture was allowed to stir for a further 3hours at which point TLC showed completion of reaction. The reaction wasquenched by pouring it onto a mixture of ice/water mixture and theresulting mixture was extracted with ethyl acetate (3×50 mL). Thecombined extracts were sequentially washed with saturated aqueous NaHCO₃(50 mL), water (50 mL), brine (50 mL) and finally dried over MgSO₄.Excess ethyl acetate was evaporated by rotary evaporator under reducedpressure and the crude product was used for the boc deprotection step toafford 8 without further purification. For boc deprotection, the crudeproduct was dissolved in a small volume of MeOH and 4M HCl in dioxane (5mL) was added slowly to the stirring solution. The reaction mixture wasstirred for 2 hours at which point TLC showed completion of reaction.Excess solvent was evaporated under vacuum to obtain a brown colouredsolid (8). The solid product was subjected to flash chromatography(n-hexane/EtOAc 9:1) to give pure 8. Yield 0.21 gm, 77%.

¹H-NMR (400 MHz, CDCl₃): δ 7.72 (1H, s), 7.69 (1H, s), 7.57 (2H, d,J=8.0 Hz), 7.46 (4H, d, J=8.0 Hz), 7.41 (2H, d, J=8.0 Hz), 7.20 (1H, d,J=2.0 Hz), 7.06 (1H, d, J=2.0 Hz, 7.02 (1H, d, J=1.6 Hz), 6.92 (1H, s).m/z (+EI) calc. for C₂₅H₂₄N₄O₃ (M)⁺ 428.48 found 429.26 ([M+H]⁺

(d)

(i) Methyl4-(4-(4-aminophenyl)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylate(9)

Two eq of EDCl and 2.5 eq of DMAP were added to a stirred solution of 6(0.45 gm, 1.2 eq) in DMF (8 mL) and the mixture was allowed to stir for30 minutes after which methyl 4-amino-1-methyl-1H-pyrrole-2-carboxylate(0.18 g, 1.18 mmol, 1.0 eq) was added. The reaction mixture was allowedto stir for a further 6 hours at which point TLC showed completion ofreaction. The reaction was quenched by pouring it onto a mixture ofice/water mixture and the resulting mixture was extracted with ethylacetate (3×150 mL). The combined extracts were sequentially washed withcitric acid (100 mL), saturated aqueous NaHCO₃ (100 mL), water (100 mL),brine (100 mL) and finally dried over MgSO₄. Excess ethyl acetate wasevaporated by rotary evaporator under reduced pressure and the crudeproduct 9a (0.58 gm, yield 90.6%) was used for the boc deprotection stepto afford 9 without further purification. For boc deprotection, 0.29 gmof 9a was dissolved in a small volume of MeOH and 4M HCl in dioxane (15mL) was added slowly to the stirring solution. The reaction mixture wasstirred for 6 hours at which point TLC showed completion of reaction.Excess solvent was evaporated under vacuum to obtain a brown colouredsolid (9). The solid product was subjected to flash chromatography(n-hexane/EtOAc 9:1) to give pure 9. Yield 0.21 gm, 95%.

(ii) Methyl4-(4-(4-(4-aminophenyl)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylate(10)

Lithium hydroxide (68 mg, 1.65 mmol, 3 eq) was added to 9a (0.25 g, 0.55mmol) in aqueous dioxane (8 ml dioxane, 4 ml water) at room temperature.The reaction mixture was stirred for 3 hours at which point TLC showedcompletion of reaction. Dioxane was evaporated under high vacuum and theresidue was diluted with water. The resulting solution was acidifiedwith 1 M citric acid followed by extraction with ethyl acetate (2×50mL). The organic layer combined and washed with brine (50 mL), driedover MgSO₄ and finally concentrated using a rotary evaporator underreduced pressure to obtain the hydrolysed acid form of 9a as a whitesolid (yield 0.23 g, 91.6%). To a stirring solution of the white solid(0.23 gm, 0.52 nmol) in DMF, 2.0 equivalent of EDCl and 2.5 Equivalentof DMAP was added. After stirring the mixture for 20 minutes,commercially available methyl 4-amino-1-methyl-1H-pyrrole-2-carboxylate(80.1 mg, 0.52 mmol, 1.0 eq) was added. The reaction mixture was allowedto stir for a further 3 hours at which point TLC showed completion ofreaction. The reaction was quenched by pouring it onto a mixture ofice/water mixture and the resulting mixture was extracted with ethylacetate (3×50 mL). The combined extracts were sequentially washed withsaturated aqueous NaHCO₃ (50 mL) and brine (50 mL) and finally driedover MgSO₄. Excess ethyl acetate was evaporated by rotary evaporatorunder reduced pressure and the crude product was used for the bocdeprotection step to afford 10. For boc deprotection, the crudeintermediate was dissolved in a small volume of MeOH and 4M HCl indioxane (5 mL) was added slowly to the stirring solution. The reactionmixture was stirred for 3 hours at which point TLC showed completion ofreaction. Excess solvent was evaporated under vacuum to obtain a browncoloured solid (10). The solid product was subjected to flashchromatography (n-hexane/EtOAc 8:2) to give pure 10. Yield 0.20 gm, 83%over 2 steps. ¹H-NMR (DMSO, 400 MHz): δ 9.72 (1H, s), 8.09 (1H, t, J=5.6Hz), 7.71 (2H, d, J=8.8 Hz), 7.49 (2H, d, J=8.8 Hz), 7.40 (1H, d, J=2.0Hz), 7.27 (1H, d, J=2.0), 7.19 (1H, d, J=2.0), 7.03 (1H, dd, J=4.0, 1.6Hz), 7.00 (1H, t, J=2.0 Hz), 6.84 (1H, d, J=2.0 Hz), 6.10 (1H, m), 3.89(3H, s). m/z (+EI) calc. for C₂₅H₂N₆O₄ (M)⁺ 474.51 found 475.35 ([M+H]⁺

(e)

Methyl4-(4-(4-aminophenyl)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-imidazole-2-carboxylate(11)

0.3 gm of boc protected 6 (0.94 mmol, 1.2 eq) was dissolved in DMF (5mL) to which 2.0 eq of EDCl and 2.5 eq of DMAP were added. The mixturewas allowed to stir for 30 minutes after which methyl4-amino-1-methyl-1H-imidazole-2-carboxylate (0.121 g, 0.79 mmol, 1.0 eq)was added. The reaction mixture was allowed to stir for a further 6hours at which point TLC showed completion of reaction. The reaction wasquenched by pouring it onto a mixture of ice/water mixture and theresulting mixture was extracted with ethyl acetate (3×150 mL). Thecombined extracts were sequentially washed saturated aqueous NaHCO₃ (50mL), water (50 mL), brine (50 mL) and finally dried over MgSO₄. Excessethyl acetate was evaporated by rotary evaporator under reduced pressureand the crude product 11a (0.48 gm) was used for the boc deprotectionstep to afford 11. For boc deprotection, the crude intermediate wasdissolved in a small volume of MeOH and 4M HCl in dioxane (5 mL) wasadded slowly to the stirring solution. The reaction mixture was stirredfor 2 hours at which point TLC showed completion of reaction. Excesssolvent was evaporated under vacuum to obtain a brown coloured solid(11). The solid product was subjected to flash chromatography(n-hexane/EtOAc 9:1) to give pure 11. Yield 0.35 gm, 81% over two steps.

¹H-NMR (DMSO, 400 MHz): 9.75 (1H, s), 8.03 (1H, s), 7.71 (2H, d, J=8.8Hz, 7.53 (1H, s), 7.52 (1H, s), 7.48 (2H, d, J=8.8 Hz), 7.42 (1H, s),7.19 (1H, d, J=2.0), 3.94 (3H, s), 3.91 (3H, s), 3.89 (3H, s). m/z (+EI)calc. for C₁₈H₁₉N₅O₃ (M)⁺ 353.38 found 354.42 ([M+H]⁺

(f)4-(10-(allyloxycarbonyl)-7-methoxy-5-oxo-11-(tetrahydro-2H-pyran-2-yloxy)-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanoicacid (13)

A 0.5 M solution of NaOH (made from 1.4135 g of NaOH) was added to asolution of 12 (Compound 18, WO 2007/039752) in dioxane at roomtemperature. The reaction mixture was allowed to stir for 4 hours atwhich point TLC showed completion of the reaction. Dioxane wasevaporated under high vacuum and the residue was diluted with water. Theresulting solution was acidified with 1 M citric acid followed byextraction with ethyl acetate (2×100 mL). The combined organic layerswere washed with brine (100 mL), dried over MgSO₄ and finallyconcentrated using a rotary evaporator under reduced pressure. Yield—8.7g, (94%), ¹H-NMR (400 MHz, CDCl₃): δ 7.2 (2H, s), 6.90 (1H, s), 6.58(1H, s), 5.85 (2H, d, J=9.2 Hz), 5.73 (2H, d, J=9.2 Hz), 5.03-5.13 (m,6H), 4.68-4.35 (m, 4H), 4.09-4.01 (m, 4H), 3.91-3.82 (m, 8H), 3.69-3.46(m, 8H), 2.60-2.55 (m, 4H), 2.18-2.00 (m, 10H), 1.76-1.55 (m, 4H),1.53-1.43 (m, 8H); ¹³C-NMR (100 MHz, CDCl3): δ 177.6, 167.6, 149.8,132.1, 131.9, 126.7, 117.3, 114.9, 110.8, 100.7, 96.0, 91.7, 88.5, 67.9,66.6, 63.6, 60.1, 56.1, 46.5, 31.1, 30.3, 28.8, 25.2, 24.1, 23.2, 20.0;EIMS m/z (+EI) calc. for C26H34N2O9 (M)⁺ 518.56 found 519.26 (M+H)⁺

(g) Methyl4-[[4-[[4-(4-aminophenyl)-1-methyl-pyrrole-2-carbonyl]amino]-1-methyl-imidazole-2-carbonyl]amino]-1-methyl-pyrrole-2-carboxylate(19)

Lithium hydroxide (40 mg, 1.65 mmol, 3 eq) was added to 11a (0.25 g,0.55 mmol) in aqueous dioxane (8 ml dioxane, 4 ml water) at roomtemperature. The reaction mixture was stirred for 3 hours at which pointTLC showed completion of reaction. Dioxane was evaporated under highvacuum and the residue was diluted with water. The resulting solutionwas acidified with 1 M citric acid followed by extraction with ethylacetate (2×50 mL). The organic layer combined and washed with brine (50mL), dried over MgSO₄ and finally concentrated using a rotary evaporatorunder reduced pressure to obtain the hydrolysed acid form of 17 as awhite solid (yield 0.235 g, 97%) which was used for the next reactionwithout any further purification. To a stirring solution 17 (0.235 gm,0.54 nmol) in DMF, 2.0 equivalent of EDCl and 2.5 Equivalent of DMAP wasadded. After stirring the mixture for 20 minutes, commercially availablemethyl 4-amino-1-methyl-1H-imidazole-2-carboxylate (100.00 mg, 0.54mmol, 1.2 eq) was added. The reaction mixture was allowed to stir for afurther 3 hours at which point TLC showed completion of reaction. Thereaction was quenched by pouring it onto a mixture of ice/water mixtureand the resulting mixture was extracted with ethyl acetate (3×50 mL).The combined extracts were sequentially washed with saturated aqueousNaHCO₃ (50 mL) and brine (50 mL) and finally dried over MgSO₄. Excessethyl acetate was evaporated by rotary evaporator under reduced pressureand the crude product was used for the boc deprotection step to afford18 which was dissolved in a small volume of MeOH and 4M HCl in dioxane(5 mL) was added slowly to the stirring solution. The reaction mixturewas stirred for 3 hours at which point TLC showed completion ofreaction. Excess solvent was evaporated under vacuum to obtain a browncoloured solid (19). The solid product was subjected to flashchromatography (n-hexane/EtOAc 8:2) to give pure 19. Yield 0.22 gm, 85%over 2 steps.

¹H-NMR (DMSO, 400 MHz): 10.09 (1H, s), δ 9.89 (1H, s), 7.78 (2H, d,J=8.8), 7.68 (1H, s), 7.49 (2H, d, J=8.64), 7.35 (1H, d, J=1.6), 7.21(1H, d, J=2.0 Hz), 7.15 (1H, d, J=2.0 Hz), 3.97 (6H, s), 3.90 (3H, s),3.84 (3H, s) m/z (+EI) calc. for C₂₃H₂₃N7O₄ (M)⁺ 461.47 found 462.17([M+H]+

Example 1

A Q² 15/16a*  5 MPB

15/16b*  8 MPB-MPB

15/16c  9 MPB-Py

15/16d 11 MPB-Im

15/16g 10 MPB-Py-Py

15/16h 19 MPB-Py-Im

*comparative examples

(a) (S)-methyl4-(4-(4-(7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(16a) (i) (11aS)-allyl7-methoxy-8-(4-(4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)phenylamino)-4-oxobutoxy)-5-oxo-11-(tetrahydro-2H-pyran-2-yloxy)-2,3,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate(15a)

A solution of Alloc-THP protected PBD acid 13 (3.72 g, 7.16 mmol, 1.2equivalent) was dissolved in DMF. EDCl (2.49 g, 13.02 mmol, 2.0 eq) andDMAP (1.989 g, 16.28 mmol, 2.5 eq) were added to the stirred solution of13 at room temperature and the mixture was allowed to stir for 30minutes after which the MPB-ester 7 (1.5 g, 6.514 mmol, 1.0 eq) wasadded. The reaction mixture was allowed to stir for a further 2 hours atwhich point TLC showed completion of reaction. The reaction was quenchedby pouring it onto a mixture of ice/water mixture and the resultingmixture was extracted with ethyl acetate (3×150 mL). The combinedextracts were sequentially washed with citric acid (200 mL), saturatedaqueous NaHCO₃ (250 mL), water (250 mL), brine (250 mL) and finallydried over MgSO₄. Excess ethyl acetate was evaporated by rotaryevaporator under reduced pressure and the crude product was purified bysilica gel flash chromatography (MeOH: CHCl₃, 20:80) to give a whitefoamy solid, 15a. Yield—4.05 g, 85.5%. (FTIR, v_(max)/cm⁻¹): 2949, 2362,1704, 1600, 1514, 1436, 1372, 1269, 1203, 1107, 1021, 964, 765. (¹H NMR,400 MHz, CDCl₃): δ 7.82 (1H, s), 7.48 (2H, m), 7.41 (1H, d, J=2.0 Hz),7.40 (1H, d, J=2.4 Hz), 7.23 (2H, d, J=8.4 Hz), 7.17 (1H, d, J=2.0 Hz),7.04 (1H, d, J=2.0 Hz), 5.93-5.65 (2H, m), 5.09-5.4.97 (m, 4H),4.68-4.32 (m, 4H), 4.15-4.10 (m, 4H), 3.94-3.82 (m, 12H), 3.68 (m, 2H),3.59-3.49 (m, 6H), 2.60-2.57 (m, 3H), 2.15-2.00 (m, 8H), 1.88-1.80 (m,2H), 1.79-1.70 (6H), 1.60-1.44 (m, 12H); (¹³C NMR, 100 MHz, CDCl₃): δ177.1, 170.5, 167.3, 161.6, 149.1, 136.3, 132.1, 131.9, 130.4, 128.9,127.1, 125.9, 125.4, 123.5, 123.1, 120.3, 117.3, 114.6, 110.8, 91.5,88.6, 68.2, 66.5, 64.3, 63.6, 60.3, 56.0, 51.1, 46.4, 36.8, 31.1, 30.9,29.1, 25.1, 24.6, 23.2, 21.0, 20.1; m/z (+EI) calc. for C₃₉H₄₆N₄O₁₀ (M)⁺730.80 found 731.67 ([M+H]*

(ii) (S)-methyl4-(4-(4-(7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(16a)

Palladium tetrakis[triphenylphosphine](5.60 mg, 4.8 μM, 0.05 equiv) wasadded to a solution of Alloc-THP-PBD conjugate 15a (70 mg, 0.097 mmol),pyrrolidine (8.36 mg, 0.117 mmol, 1.2 eq) and triphenylphosphine (8.62mg, 0.25 equiv) in DCM (5 mL). The reaction mixture was stirred at roomtemperature for 2 hours at which point TLC showed completion ofreaction. Excess DCM was removed by rotary evaporation under reducedpressure and the resulting residue dried in vacuo to remove pyrrolidone.The product was purified by column chromatography (eluted with n-hexane65%%, EtOAc 35%) to give the product as a yellowish solid, 3.37 (40 mg,77%). [α]^(22.7) _(D)+165° (c=0.046, CHCl3); IR (FTIR, v^(max)/cm⁻¹):3297, 2944, 2358, 1701, 1598, 1567, 1508, 1442, 1374, 1264, 1212, 1181,1106, 1072, 824, 730; ¹H-NMR (500 MHz, CDCl₃): δ 7.68 (1H, s), 7.65 (1H,d, J=4.5 Hz, H-11), 7.52 (1H, s, H-6), 7.46 (2H, dd, J=8.4, 2.0 Hz,2Ar—H), 7.40 (2H, dd, J=8.4, 2.0 Hz, 2Ar—H), 7.16 (1H, d, J=2.0 Hz,Py-H), 7.03 (1H, d, J=1.6 Hz, Py-H), 6.82 (1H, s, H-9), 4.12-4.20 (2H,m, CH₂ side chain linker), 3.94 (3H, s, N—CH₃), 3.88 (3H, s, O—CH₃),3.68-3.71 (1H, m, H-11a), 3.50-3.60 (2H, m, H2-3), 2.58-2.62 (2H, m,CH₂), 2.26-2.31 (4H, m, CH₂), 1.50-1.54 (2H, m, CH₂); ¹³C-NMR (125 MHz,CDCl₃): δ 164.5, 162.4, 161.6, 150.5, 147.8, 140.7, 125.9, 125.5 (2C),123.6, 123.1, 120.3, 114.6, 111.8, 111.0, 94.4 (2C), 68.0, 63.7, 56.1,53.7, 51.0, 46.6, 36.8, 31.9, 29.6, 25.2, 24.8, 24.1, 20.2; HRMS m/z(+EI) calc. for C₃₀H₃₂N₄O (M+H)⁺ 545.2400 found 545.2422 (M+H)⁺, δ 4 ppm

Compounds 15b-d, g, h and 16b-d, g, h were made in an analogous manner,reacting compound A with 13, followed by deprotection.

(b) (S)-methyl4-(4-(4-(4-(4-(7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(16b)

[α]^(22.7) _(D)+134° (c=0.038, CHCl₃); IR (FTIR, v_(max)/cm⁻¹): 3850.89,3732, 3619, 2443, 2354, 2228, 2169, 2091, 1971, 1859, 1729, 1679, 1521,1265, 734, 629; ¹H-NMR (500 MHz, CDCl₃): δ 7.72 (1H, s, NH), 7.69 (1H,s, NH), 7.66 (1H, d, J=4.0 Hz, H-11), 7.57 (2H, d, J=8.0 Hz, 2Ar—H),7.53 (1H, s, H-6), 7.46 (4H, d, J=8.0 Hz, 4Ar—H), 7.41 (2H, d, J=8.0 Hz,2Ar—H), 7.20 (1H, d, J=2.0 Hz, Py-H), 7.06 (1H, d, J=2.0 Hz, Py-H), 7.02(1H, d, J=1.6 Hz, Py-H), 6.92 (1H, s, Py-H), 6.84 (1H, s, H-9),4.12-4.20 (2H, m, CH₂ side chain linker), 4.00 (3H, s, N—CH₃), 3.96 (3H,s, N—CH₃), 3.88 (3H, s, O—CH₃), 3.84 (3H, s, O—CH₃), 3.70-3.73 (1H, m,H-11a), 3.55-3.61 (2H, m, H₂-3), 2.58-2.62 (2H, m, CH₂), 2.29-2.31 (2H,m, CH₂), 1.93-2.06 (4H, m, CH₂); ¹³C-NMR (125 MHz, CDCl₃): δ 164.5,162.4, 161.7, 150.7, 147.3, 139.2, 126.0, 125.6, 125.4 (2C), 125.2 (2C),123.0, 120.4 (2C), 114.6 (2C), 111.4, 94.6 (2C), 68.3, 63.7, 56.1, 51.6(2C), 41.0, 36.9, 31.9, 29.6, 25.2, 24.2, 24.1, 20.2; HRMS m/z (+EI)calc. for C₄₂H₄₂N₆O₇ (M+H)⁺ 743.3193 found 743.3193 ([M+H]⁺, δ 0.3 ppm)

(c) (S)-methyl4-(4-(4-(4-(7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylate(16c)

[α]^(22.7) _(D)+128° (c=0.037, CHCl₃); IR (FTIR, v_(max)/cm⁻¹): 3321,2237, 2107, 2041, 1967, 1860, 1685, 1517, 1435, 1254, 1180, 1118, 749,722, 696, 667; ¹H-NMR (500 MHz, CDCl₃): δ 7.98 (1H, s, NH), 7.88 (1H, s,NH), 7.68 (1H, s, H-6), 7.65 (1H, d, J=4.0 Hz, H-11), 7.64 (2H, d, J=8.0Hz, 2Ar—H), 7.54 (1H, d, J=1.6 Hz, Py-H), 7.52 (1H, d, J=1.6 Hz, Py-H),7.45 (1H, d, J=2.0 Hz, Py-H), 7.33 (2H, d, J=8.0 Hz, 2Ar—H), 6.97 (1H,s, Py-H), 6.89 (1H, s, H-9), 4.08-4.18 (2H, m, CH₂), 3.97 (3H, s,N—CH₃), 3.89 (3H, s, N—CH₃), 3.84 (3H, s, O—CH₃), 3.79 (3H, s, O—CH₃),3.66-3.70 (1H, m, H-11a), 3.55-3.60 (2H, m, H₂-3), 2.56-2.61 (2H, m,CH₂), 2.23-2.32 (4H, m, CH₂), 2.00-2.05 (2H, m); ¹³C-NMR (125 MHz,CDCl₃): δ 162.5, 161.6, 159.1, 150.4, 147.7, 138.4, 132.8, 132.1 131.9(2C), 128.6, 128.4 (2C), 125.4 (2C), 124.8, 123.0, 121.0, 120.4 (2C),116.2, 114.6 (2C), 109.9, 94.2, 67.4, 63.6, 57.1, 53.7, 51.1, 46.7,36.9, 36.7, 34.0, 29.6, 24.2; HRMS m/z (+EI) calc. for C₃₆H₃₈N₆O₇ (M)⁺667.2880 found 667.2881 ([M+H]⁺, δ 0.1 ppm)

(d) (S)-ethyl4-(4-(4-(4-(7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-imidazole-2-carboxylate(16d)

[α]^(22.7) _(D)+122° (c=0.028, CHCl₃), IR (FTIR, v_(max)/cm⁻¹): 3324,2355, 2157, 2109, 2032, 1913, 1600, 1533, 1465, 1262, 1179, 1109, 751;¹H-NMR (500 MHz, CDCl₃): δ 8.47 (1H, s, NH), 7.72 (1H, s, NH), 7.66 (1H,d, J=4.0 Hz, H-11), 7.55 (1H, s, H-6), 7.52 (1H, d, J=2.0, Py-H), 7.49(2H, d, J=8.0 Hz, 2Ar—H), 7.37 (2H, d, J=8.0 Hz, 2Ar—H), 7.16 (1H, d,J=1.6 Hz, Py-H), 7.03 (1H, s, Im-H), 6.91 (1H, s, H-9), 4.39-4.43 (2H,m, O—CH₂) 4.13-4.22 (2H, m, CH₂), 4.01 (3H, s, N—CH₃), 3.99 (3H, s,N—CH₃), 3.83 (3H, s, O—CH₃), 3.68-3.72 (1H, m, H-11a), 3.55-3.60 (2H, m,H₂-3), 2.58-2.63 (2H, m, CH₂), 2.24-2.32 (4H, m, CH₂), 2.00-2.07 (2H, m,H₂-1), 141-1.45 (3H, m, CH₃); ¹³C-NMR (125 MHz, CDCl₃): δ 163.1, 162.5,158.7, 150.4, 147.7, 140.7, 137.2, 131.5, 125.6 (2C), 125.4 (2C), 123.8,123.0, 120.4 (2C), 114.5, 111.6, 110.8, 109.9, 100.0, 67.4, 61.5, 56.1,53.7, 51.1, 46.7, 37.0, 36.0, 34.0, 29.6, 24.8, 24.2, 14.4; HRMS m/z(+EI) calc. for C₃₆H₃₉N₇O₇ (M)⁺ 682.2989 found 682.2986 ([M+H]⁺, δ−0.4ppm).

(e) (S)-methyl4-(4-(4-(4-(4-(7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylate(16g)

[α]^(22.7) _(D)+149° (c=0.054, CHCl₃); IR (FTIR, v_(max)/cm⁻¹): 3310,2947, 2358, 2168, 2153, 2132, 2070, 2011, 1989, 1651, 1538, 1434, 1402,1257, 1107, 753; ¹H-NMR (400 MHz, CDCl₃): b 8.02 (1H, s, NH), 7.88 (1H,d, J=5.2 Hz, H-11), 7.68 (1H, s, H-6), 7.67 (1H, d, J=1.6 Hz, Py-H),7.64 (1H, d, J=1.6 Hz, Py-H), 7.53 (2H, d, J=8.0 Hz, 2Ar—H), 7.45 (1H,d, J=1.6 Hz, Py-H), 7.31 (2H, d, J=8.0 Hz, 2Ar—H), 7.20 (1H, s, Py-H),6.96 (1H, s, Py-H), 6.89 (1H, bs, NH), 6.81 (1H, s, H-9), 6.78 (1H, d,J=1.6 Hz, Py-H), 6.71 (1H, bs, NH), 4.11-4.16 (2H, m, CH₂), 3.97 (3H, s,N—CH₃), 3.92 (3H, s, N—CH₃), 3.88 (3H, s, N—CH₃), 3.84 (3H, s, N—CH₃),3.79 (3H, s, O—CH₃), 3.68-3.71 (1H, m, H-11a), 3.55-3.60 (2H, m, H₂-3),2.56-2.61 (2H, m, CH₂), 2.22-2.28 (4H, m, CH₂), 1.99-2.04 (2H, m); HRMSm/z (+EI) calc. for C₄₁H₄₃N₉O₈ (M)⁺ 790.3313 found 790.3314 [M+H]⁺, b0.1 ppm.

(f) (S)-methyl4-(4-(4-(4-(4-(7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-imidazole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylate(16h)

[α]^(22.7) _(D)+142° (c=0.043, CHCl₃); IR (v_(max)/cm⁻¹): 3408, 2358,2168, 2148, 2019, 1978, 1938, 1718, 1534, 1260, 1118, 757; ¹H-NMR (500MHz, CDCl₃): δ 8.72 (1H, s, NH), 8.12 (1H, s, NH), 7.71 (1H, s), 7.65(1H, d, J=4.4 Hz), 7.53 (1H, s), 7.48 (2H, d, J=8.0 Hz), 7.47 (1H, s),7.42 (2H, d, J=1.6 Hz), 7.40 (2H, d, J=8.0 Hz), 7.03 (1H, d, J=1.6 Hz),6.95 (1H, s), 6.82 (1H, s), 6.81 (1H, d, J=1.6 Hz), 4.12-4.21 (2H, m),4.07 (3H, s), 4.00 (3H, s), 3.91 (3H, s), 3.89 (3H, s), 3.81 (3H, s),3.69-3.72 (1H, m), 3.55-3.61 (2H, m), 2.58-2.63 (2H, m), 2.26-2.32 (4H,m, CH₂), 2.02-2.07 (2H, m); HRMS (EI, m/z): Calc. for C₄₁H₄₃N₉O₈ (MH⁺):790.3313. Found, 790.3314.

Example 2

Q¹ 14a

14b

Q² 15/16e Py-MPB

15/16f Im-MPB

(a)

(i)4-(4-(((11S,11aS)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxylicacid (14a)

A solution of Alloc-THP protected PBD acid 13 (1.85 g, 3.57 mmol, 1.2equivalent) was dissolved in DMF. EDCl (1.24 g, 6.48 mmol, 2.0 eq) andDMAP (0.99 g, 8.1 mmol, 2.5 eq) were added to the stirred solution of 13at room temperature and the mixture was allowed to stir for 30 minutesafter which methyl 4-amino-1-methyl-1H-pyrrole-2-carboxylate (0.5 g,3.243 mmol, 1.0 eq) was added. The reaction mixture was allowed to stirfor a further 6 hours at which point TLC showed completion of reaction.The reaction was quenched by pouring it onto a mixture of ice/watermixture and the resulting mixture was extracted with ethyl acetate(3×150 mL). The combined extracts were sequentially washed with citricacid (200 mL), saturated aqueous NaHCO₃ (250 mL), water (250 mL), brine(250 mL) and finally dried over MgSO₄. Excess ethyl acetate wasevaporated by rotary evaporator under reduced pressure and the crudeproduct (1.88 gm) was used for hydrolysis reaction to afford 14a. Forhydrolysis, Lithium hydroxide (0.24 g, 5.71 mmol, 3 eq) was added to thecrude product (1.88 g, 2.87 mmol) in aqueous dioxane (75 ml dioxane,11.5 ml water) at room temperature. The reaction mixture was stirred for3 hours at which point TLC showed completion of reaction. Dioxane wasevaporated under high vacuum and the residue was diluted with water. Theresulting solution was acidified with 1 M citric acid followed byextraction with ethyl acetate (2×100 mL). The organic layer combined andwashed with brine (100 mL), dried over MgSO₄ and finally concentratedusing a rotary evaporator under reduced pressure to obtain 14a as awhite solid (yield, 1.68 gm, 74.0% over 2 steps). ¹H-NMR δ 9.09 (1H, s,NH), 7.39 (1H, d, J=2.0 Hz), 7.14 (1H, s, H-6), 7.12 (1H, s, H-6), 6.96(1H, s, H-9), 6.76 (1H, d, J=2.0 Hz, Py-H), 5.86-5.75 (2H, m, H-11),5.13-4.84 (3H, m), 4.61-4.21 (2H, m), 4.06-3.88 (3H, m, side-chain H-1,pyran H-6), 3.87 (3H, s, O/NCH3), 3.87 (3H, s, O/NCH3), 3.86 (3H, s),3.53-3.44 (3H, m), 2.55-2.45 (2H, m), 2.13-1.88 (6H, m), 1.70-1.39 (6H).m/z (+EI) calc. for C₃₂H₄₀N₄O₁₀ (M)⁺ 640.68 found 641.57 ([M+H]⁺

(ii) (11S,11aS)-allyl7-methoxy-8-(4-((5-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(15e)

A solution of Alloc-THP protected PBD-Py acid 14a (150 mg, 0.23 mmol,1.0 equivalent) was dissolved in DMF. EDCl (2.49 g, 13.02 mmol, 2.0 eq)and DMAP (1.989 g, 16.28 mmol, 2.5 eq) were added to the stirredsolution of 13 at room temperature and the mixture was allowed to stirfor 30 minutes after which the MPB-ester 7 (67.83 mg, 0.29 mmol, 1.25eq) was added. The reaction mixture was allowed to stir for a further 3hour at which point TLC showed completion of reaction. The reaction wasquenched by pouring it onto a mixture of ice/water mixture and theresulting mixture was extracted with ethyl acetate (3×150 mL). Thecombined extracts were sequentially washed with citric acid (50 mL),saturated aqueous NaHCO₃ (50 mL), water (50 mL), brine (50 mL) andfinally dried over MgSO₄. Excess ethyl acetate was evaporated by rotaryevaporator under reduced pressure and the crude product was directlyused in the next step without further purification. m/z (+EI) calc. forC₄₅H₅₂N₆O₁₁ (M)⁺ 852.93 found 854.87 ([M+H]

(iii) (S)-methyl4-(4-(4-(4-(7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(16e)

Palladium tetrakis[triphenylphosphine](12.17 mg, 10.5 μM, 0.05 equiv)was added to a solution of Alloc-THP-PBD conjugate 15e (179 mg, 0.21mmol), pyrrolidine (17.91 mg, 0.25 mmol, 1.2 eq) and triphenylphosphine(13.81 mg, 0.25 equiv) in DCM (5 mL). The reaction mixture was stirredat room temperature for 2 h at which point TLC showed completion ofreaction. Excess DCM was removed by rotary evaporation under reducedpressure and the resulting residue dried in vacuo to remove pyrrolidone.The product was purified by high performance liquid chromatography(eluted with acetone:water gradient with 1% TFA) to give the product asa light yellowish solid, 16e (48 mg, 34% after HPLC purification).

[α]^(22.7) _(D)+197° (c=0.052, CHCl₃), IR (FTIR, v_(max)/cm⁻¹): 3330,2360, 2214, 2180, 2041, 2020, 1999, 1967, 1698, 1517, 1438, 1265, 1180,1119, 756, 722, 696, 667, 630; ¹H-NMR (500 MHz, CDCl₃): δ 7.77 (1H, s,NH), 7.68 (1H, s, H-6), 7.67 (2H, d, J=8.0 Hz, 2Ar—H), 7.64 (1H, d,J=5.0 Hz, H-11), 7.55 (2H, d, J=8.0 Hz, 2Ar—H), 7.47 (1H, d, J=2.0 Hz,Py-H), 7.43 (1H, s, NH), 7.18 (1H, d, J=2.0 Hz, Py-H), 7.09 (1H, d,J=2.0 Hz, Py-H), 7.05, (1H, d, J=2.0 Hz, Py-H), 6.83 (1H, s, H-9),4.09-4.16 (2H, m, CH₂), 3.95 (3H, s, N—CH₃), 3.90 (6H, s, N—CH₃, O—CH₃),3.84 (3H, s, O—CH₃), 3.67-3.71 (1H, m, H-11a), 3.54-3.57 (2H, m, Hr₂-3),2.53-2.56 (2H, m, CH₂), 2.23-2.30 (4H, m, CH₂), 2.00-2.05 (2H, m);¹³C-NMR (125 MHz, CDCl₃): δ 169.8, 164.5, 162.6, 161.6, 159.5, 150.7,147.9, 140.8, 133.1, 132.2, 132.1, 131.9, 131.7, 128.5, 128.4, 125.9,125.5, 123.7, 123.1, 121.5, 120.7, 120.4, 119.7, 114.7, 112.0, 111.4,103.9, 68.1, 56.2, 53.7, 51.0, 46.7, 36.8, 33.2, 29.6, 25.1, 24.1; HRMSm/z (+EI) calc. for C₃₆H₃₈N₆O₇ (M)⁺ 667.2880 found 667.2882 ([M+H]⁺, δ0.3 ppm).

Compounds 14b, 15f and 16f, were made in an analogous manner, reactingcompound 13 with the imidazolyl building block, followed by reactionwith the MPB building block, and finally by deprotection.

(b) (S)-methyl4-(4-(4-(4-(7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(16f)

[α]^(22.7) _(D)+188 (c=0.052, CHCl₃), IR (FTIR, v_(max)/cm⁻¹): 3301,2169, 2136, 2018, 1978, 1937, 1680, 1564, 1518, 1439, 1265, 1181, 1108,750, 722; ¹H-NMR (500 MHz, CDCl₃): δ 8.90 (1H, s, NH), 7.98 (1H, s, NH),7.67 (1H, s, H-6), 7.63 (1H, d, J=4.4 Hz, H-11), 7.59 (2H, d, J=8.4 Hz,2Ar—H), 7.46 (2H, d, J=8.4 Hz, 2Ar—H), 7.42 (1H, s, Im-H), 7.19 (1H, d,J=2.0 Hz, Py-H), 7.06 (1H, d, J=1.6 Hz, Py-H), 6.83 (1H, s, H-9),4.10-4.22 (2H, m, CH₂), 4.07 (3H, s, N—CH₃), 3.96 (6H, s, N—CH₃, O—CH₃),3.84 (3H, s, O—CH₃), 3.67-3.70 (1H, m, H-11a), 3.54-3.58 (2H, m, H₂-3),2.57-2.67 (2H, m, CH₂), 2.26-2.31 (4H, m, CH₂), 1.98-2.05 (2H, m);¹³C-NMR (125 MHz, CDCl₃): δ 169.5, 164.5, 162.5, 161.6, 156.4, 150.4,147.8, 135.6, 132.1, 131.9 (2C), 128.5, 128.4, 126.0, 125.6, 123.5,123.1, 121.5, 119.7, 114.6 (2C), 111.6, 111.0, 67.7, 56.1, 53.7, 51.1,46.6, 36.9, 35.8, 33.9, 29.6, 24.7, 24.1; HRMS m/z (+EI) calc. forC₃₅H₃₇N₇O₇ (M)⁺ 668.2833 found 668.2838 [M+H]⁺, δ 0.5 ppm.

Example 3

The cytotoxicity potential of the compounds of the invention 16c-h wascompared with the comparative compounds 16a, 16b and GWL-78 in a numberof tumour cell lines and the non-cancer cell line W138 after 96 hoursexposure using the MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)colorimetric assay, as described below.

A panel of several types of human cancer cell lines including epidermoid(A431), lung (A549), ovarian (A2780) and breast (MCF7 and MDAMB-231), aswell as the non-tumour cell line WI38, were used to determine thecytotoxicity of the compounds. The cells were grown in normal conditionsat 37° C. under a 5% CO₂ humidified atmosphere, either in Dulbecco'sModified Eagle Medium or Modified Eagle Medium (depending on the cellline), supplemented with 10% fetal bovine serum (Biosera, UK), 1%L-glutamine, 1% non-essential amino acids and 0.05% hydrocortisone(Gibco, Invitrogen, USA). Cells were then seeded into 96-well plates ina total volume of 160 μl, and allowed to reach a 30-40% degree ofconfluence before starting the experiment. The ligands were dissolved 10in sterilized ultrapure water at a maximum concentration of 100 μM, andserial decimal dilutions prepared. These were added to the cells in avolume of 40 μl. After 96 hours of continuous exposure to each ligand,the cytotoxicity was determined by the MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)(Lancaster Synthesis Ltd, UK) colorimetric assay (Skehan P, S. R., etal., Journal of the National Cancer Institute 1990, 82, 1107).Absorbance was quantified by spectrophotometry at A=570 nm (ELx808,Bio-Tek Instruments, Inc., USA). IC₅₀ values were calculated by adose-response analysis using the Origin 6.0™ software.

IC₅₀ (nM) Compound A431 A549 A2780 MCF7 MDAMB-231 Mia Paca 2 WI38 16a*2.31 7.50 1.87 1.91 2.11 1.2 159.9 16b* 2.91 0.54 0.56 0.90 0.46 0.35158.7 16c 4.60 0.019 0.96 1.70 2.70 0.34 425.9 I6d 0.19 0.47 0.15 0.370.45 0.11 1240 16e 0.0056 0.056 0.013 0.00002 0.000065 0.0013 473.8 16f0.018 0.034 0.021 0.00002 0.00018 0.0021 129.2 16g 0.86 2.3 0.24 0.310.59 0.31 41.3 16h 03064 0.45 ND 0.075 0.015 0.25 65.6 GWL-78* 0.55 6.10.57 0.32 0.12 2.4 41.3 *comparative compounds

The cytotoxicity potential of the compounds of the invention 16c-h wasalso compared with the comparative compounds 16a, 16b and GWL-78 in aprimary CLL cell lines using the Annexin V assay (van Engeland, M, etal., Cytometry 1998, 31, 1-9), as described below.

Freshly isolated peripheral blood CLL cells (1×10⁸ mL⁻¹) were culturedin RPMI medium (Invitrogen, Paisley, U.K.) supplemented with 100 U/mLpenicillin, 100 mg/mL streptomycin, and 10% fetal calf serum. The cellswere incubated at 37° C. in a humidified 5% CO₂ atmosphere in thepresence of each compound. All compounds were dissolved in DMSO and wereevaluated in serial dilutions against the primary CLL cells. Inaddition, control cultures were carried out to which no drug was added.The cytotoxic effects of the compounds were quantified using an annexinV/propidium iodide flow cytometry assay (Bender Medsystems, Vienna,Austria). All assays were performed in duplicate, and LD₅₀ values werecalculated from sigmoidal dose-response curves using the Prism 6.0software (Graphpad Software Inc., San Diego, Calif.). The sigmoidaldose-response curves were derived by plotting log [compoundconcentration]against the percentage apoptosis induced by thatconcentration. A wide range of concentrations were used to establish thebiologically active range for each individual compound.

Compound IC₅₀ (nM) 16a* 6.2 16b* 4.7 16c 2.1 I6d 3.0 16e 0.098 16f 0.1716g 0.96 16h 0.037 GWL-78* 1.3

Example 4

16e and 16f were tested in in vivo xenograft studies in mouse models ofboth breast and pancreatic cancer.

Initially a small scale study was carried out to determine the MTD(maximum tolerated dose) in Swiss-Webster mice using intraperitonealdosing (IP). The compounds were generally well tolerated without anysigns of toxicity. However, a minor weight loss was observed at a doselevel of 400 μg/Kg/day dose level for 16e (FIG. 1A). Some insignificantweight loss for 16f was also observed at the same dose level. A repeatMTD experiment using only 16f at 350 μg/Kg/day did not show any weightloss or other signs of toxicity (FIG. 1B). As 16f provided a marginallybetter toxicity profile in the MTD study, it was decided to carry outmore extensive in vivo tumour xenograft studies on this molecule.

ER-Negative MDA MB 231 Breast Cancer Xenograft Study

In vivo studies of the activity of 16f were carried out in anER-negative MDA MB 231 breast cancer xenograft in a mouse model. Thehuman breast cancer cell line MDA-MB-231 (5×10⁸ cells) was employed toestablish xenografts in the flanks of female MF1 nude mice, 2-3 monthsold and weighing 20-25 g. Subsequent passaging was by the subcutaneousimplantation of small tumour pieces (approx 1 mm³) into the flank. Whenthe tumours reached approx. 0.06 cm³ (three weeks post implantation)they were divided into 3 groups of 4 mice. The drug treated groups wereadministered with an IV dose (in DMSO) of either 250 μg/Kg/day or 300μg/Kg/day for 5 consecutive days followed by two drug free days for 3weeks, followed by 2 consecutive days in week 4 at which point thedosage was stopped. As shown in FIG. 2, 16f produced prominent in vivoantitumour activity compared to control mice (▴) at both 250 μg/Kg (♦)and 300 μg/Kg (▪) dose levels without any signs of toxicity (FIG. 2,where the arrow shows the last injected dose). The tumour did notre-grow up to 3 weeks after administration of the last IV dose in thecase of the 300 μg/Kg dose level.

Mia Paca 2 Pancreatic Cancer Xenograft in Mouse Model

An in vivo study of 16f was carried out in a pancreatic cancer xenograftmouse model, in a similar manner to the above. The drug (16f) treatedgroup was administered an IV dose of 300 μg/Kg/day for 5 consecutivedays followed by 2 drug free days, and the cycle was continued for 3weeks. 16f produced prominent antitumour activity compared to controlmice at the 300 μg/Kg dose level without any signs of toxicity (FIG. 3:♦300 μg/Kg 16f; ▪ control). The inability of the tumour to grow backimmediately after withdrawing drug was notable, and no growth wasobserved up to 21 days after the last administered dose. Cross-sectionsfrom the tumour and control tissues were subjected toimmunohistochemical staining, and the findings were consistent with NFκBinhibition in the experimental animals compared to controls.

Example 5

16f was evaluated in a commercial Transcription Factor ActivationProfiling Array Assay™ (Signosis) using the HeLa cell line. In thisassay the activities of 48 transcription factors could be monitoredsimultaneously using a collection of biotin-labeled DNA probes based onthe consensus sequences of individual transcription factor DNA-bindingsites. The top five transcription factors whose activities were at least30% down-regulated by 7h at a concentration of 10 nM for 4 hours were:NFAT, EGR, NF-κB, SMAD and OCT-4. The activity of NF-κB was reduced byalmost 50%.

Based on the hypothesis that 16f may down-regulate the expression ofNF-κB-dependent genes (e.g., IκB, BCL2, BCLX_(L)) by binding to thecognate DNA sequence of NF-κB, thereby blocking interaction of thetranscription factor protein and inhibiting transcription of a number ofgenes, it was decided to explore this possibility in CLL cells in whichNF-κB signaling is known to be active and closely correlates with theinitiation and progression of malignancy. Using levels of phosphorylatedIκB and p65 as surrogates for NF-κB activity in comparison to the Actinprotein as a control, Western Blotting indicated that, after 24 hoursincubation, 16f caused a significant suppression of phosphorylated IκBαat concentrations down to 0.1 nM, with only a marginal effect onphosphorylated-p65.

1. A compound of formula I:

or a salt or solvate thereof, wherein: the dotted double bond indicatesthe presence of a single or double bond between C2 and C3; R² isselected from —H, —OH, ═O, ═CH₂, —CN, —R, OR, halo, dihalo, ═CHR,═CHRR′, —O—SO₂—R, CO₂R and COR; R⁷ is selected from H, R, OH, OR, SH,SR, NH₂, NHR, NRR′, nitro, Me₃Sn and halo; where R and R¹ areindependently selected from optionally substituted C₁₋₇ alkyl, C₃₋₂₀heterocyclyl and C₅₋₂0 aryl groups; R¹⁰ and R¹¹ either together form adouble bond, or are selected from H and QR^(Q) respectively, where Q isselected from O, S and NH and R^(Q) is H or C₁₋₇ alkyl or H and SO_(x)M,where x is 2 or 3, and M is a monovalent pharmaceutically acceptablecation; A is either:

where X and Y are selected from: CH and NMe; COH and NMe; CH and S; Nand NMe; N and S; B is either a single bond or

Where X and Y are as defined above; and R¹ is C₁₋₄ alkyl.
 2. A compoundaccording to claim 1, where in group A, X and Y are selected from CH andNMe; CH and S; N and NMe; and N and S.
 3. A compound according to claim2, where in group A, X and Y are selected from CH and NMe; and N andNMe.
 4. A compound according to claim 1, wherein B is a single bond. 5.A compound according to claim 1, wherein B is B1, and X and Y in B1, areselected from CH and NMe; CH and S; N and NMe; and N and S.
 6. Acompound according to claim 5, where in group B1, X and Y are selectedfrom CH and NMe; and N and NMe.
 7. A compound according to claim 1,wherein R⁷ is selected from H, OR, SH, SR, NH₂, NHR, NRR′, and halo. 8.A compound according to claim 7, wherein R⁷ is selected from H and OR.9. A compound according to claim 8, wherein R⁷ is OR^(7A), where R^(7A)is optionally substituted C₁₋₇ alkyl.
 10. A compound according to claim9, wherein R^(7A) is selected from Me, CH₂Ph and allyl.
 11. A compoundaccording to claim 1, wherein R¹⁰ and R¹¹ form a double bond together.12. A compound according to claim 1, wherein R¹⁰ is H and R¹¹ is OR^(Q).13. A compound according to claim 12, wherein R^(Q) is selected from Hor Me.
 14. A compound according to claim 1, wherein R¹⁰ is H and R¹¹ isSO₃M.
 15. A compound according to claim 14, wherein M is Na⁺.
 16. Acompound according to claim 1, wherein R¹ is C₁₋₂ alkyl.
 17. A compoundaccording to claim 16, wherein R¹ is methyl.
 18. A compound according toclaim 1, wherein R² is selected from —H, —OH, ═O, ═CH₂, —CN, —R, —OR,═CHR, ═CRR′, —O—SO₂—R, —CO₂R and —COR.
 19. A compound according to claim18, wherein R² is selected from —H, ═CH₂, —R, ═CHR, and ═CRR′.
 20. Acompound according to claim 18, wherein R² is of the configuration C1:


21. A compound according to claim 1, wherein R² is optionallysubstituted C₅₋₂₀ aryl.
 22. A compound according to claim 21, wherein R²is selected from optionally substituted phenyl, optionally substitutednapthyl, optionally substituted pyridyl, optionally substitutedquinolinyl or isoquinolinyl
 23. A compound according to claim 21,wherein R² group bears one to three substituent groups.
 24. A compoundaccording to claim 21, wherein the optional substituents are selectedfrom methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene,methyl-piperazinyl, morpholino and methyl-thienyl.
 25. A compoundaccording to claim 21, wherein R² is selected from 4-methoxy-phenyl,3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4-fluoro-phenyl,4-chloro-phenyl, 3,4-bisoxymethylene-phenyl, 4-methylthienyl,4-cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl,isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl,methoxynaphthyl, and naphthyl.
 26. A compound according to claim 1,wherein R² is selected from: (a) C₁₋₅ saturated aliphatic alkyl; (b)C₃₋₆ saturated cycloalkyl; (c)

wherein each of R²¹, R²² and R²³ are independently selected from H, C₁₋₃saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where thetotal number of carbon atoms in the R¹² group is no more than 5; (d)

wherein one of R^(25a) and R^(25b) is H and the other is selected from:phenyl, which phenyl is optionally substituted by a group selected fromhalo methyl, methoxy; pyridyl; and thiophenyl; and (e)

where R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted bya group selected from halo methyl, methoxy; pyridyl; and thiophenyl. 27.A compound according to claim 26, wherein R² is selected from methyl,ethyl, propyl, butyl or pentyl.
 28. A compound according to claim 26,wherein R² is selected from cyclopropyl, cyclobutyl, cyciopentyl andcyclohexyl.
 29. A compound according to claim 26, wherein R² is

and the total number of carbon atoms in the R² group is no more than 4.30. A compound according to claim 26, wherein one of R²¹, R²² and R²³ isH, with the other two groups being selected from H, C₁₋₃ saturatedalkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl.
 31. A compoundaccording to claim 26, wherein two of R²¹, R²² and R²³ are H, with theother group being selected from H, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl,C₂₋₃ alkynyl and cyclopropyl.
 32. A compound according to claim 30,wherein the groups that are not H are selected from methyl and ethyl.33. A compound according to claim 26, wherein R² is

and the group which is not H is optionally substituted phenyl.
 34. Acompound according to claim 26, wherein R² is

and R²⁴ is selected from H and methyl.
 35. A compound according to claim1, wherein if R² is selected from any of the following groups: —H, —OH,—CN, —R, —OR, halo, —O—SO₂—R, —CO₂R and —COR, there is a double bondbetween C2 and C3.
 36. A compound according to claim 1, wherein if R² isselected from ═O, ═CH₂, ═CHR, ═CHRR′, there is a single bond between C2and C3.
 37. A compound according to claim 1, wherein there is no doublebond between C2 and C3 and R² is H.
 38. A pharmaceutical compositioncomprising a compound according to claim 1, and a pharmaceuticallyacceptable carrier or diluent.
 39. A compound according to claim 1 foruse in a method of therapy.
 40. The use of a compound according to claim1 in the manufacture of a medicament for the treatment of aproliferative disease.
 41. A compound according to claim 1 for use in amethod of treatment of a proliferative disease.
 42. A method oftreatment of a patient suffering from a proliferative disease,comprising administering to said patient a therapeutically acceptableamount of a compound according to claim 1.